Lasting Retinal Injury in a Mouse Model of Blast-Induced Trauma
2017; Elsevier BV; Volume: 187; Issue: 7 Linguagem: Inglês
10.1016/j.ajpath.2017.03.005
ISSN1525-2191
AutoresNajiba Mammadova, Shivani Ghaisas, Gary Zenitsky, Donald S. Sakaguchi, Anumantha G. Kanthasamy, Justin J. Greenlee, M. Heather West Greenlee,
Tópico(s)Traumatic Ocular and Foreign Body Injuries
ResumoTraumatic brain injury due to blast exposure is currently the most prevalent of war injuries. Although secondary ocular blast injuries due to flying debris are more common, primary ocular blast exposure resulting from blast wave pressure has been reported among survivors of explosions, but with limited understanding of the resulting retinal pathologies. Using a compressed air-driven shock tube system, adult male and female C57BL/6 mice were exposed to blast wave pressure of 300 kPa (43.5 psi) per day for 3 successive days, and euthanized 30 days after injury. We assessed retinal tissues using immunofluorescence for glial fibrillary acidic protein, microglia-specific proteins Iba1 and CD68, and phosphorylated tau (AT-270 pThr181 and AT-180 pThr231). Primary blast wave pressure resulted in activation of Müller glia, loss of photoreceptor cells, and an increase in phosphorylated tau in retinal neurons and glia. We found that 300-kPa blasts yielded no detectable cognitive or motor deficits, and no neurochemical or biochemical evidence of injury in the striatum or prefrontal cortex, respectively. These changes were detected 30 days after blast exposure, suggesting the possibility of long-lasting retinal injury and neuronal inflammation after primary blast exposure. Traumatic brain injury due to blast exposure is currently the most prevalent of war injuries. Although secondary ocular blast injuries due to flying debris are more common, primary ocular blast exposure resulting from blast wave pressure has been reported among survivors of explosions, but with limited understanding of the resulting retinal pathologies. Using a compressed air-driven shock tube system, adult male and female C57BL/6 mice were exposed to blast wave pressure of 300 kPa (43.5 psi) per day for 3 successive days, and euthanized 30 days after injury. We assessed retinal tissues using immunofluorescence for glial fibrillary acidic protein, microglia-specific proteins Iba1 and CD68, and phosphorylated tau (AT-270 pThr181 and AT-180 pThr231). Primary blast wave pressure resulted in activation of Müller glia, loss of photoreceptor cells, and an increase in phosphorylated tau in retinal neurons and glia. We found that 300-kPa blasts yielded no detectable cognitive or motor deficits, and no neurochemical or biochemical evidence of injury in the striatum or prefrontal cortex, respectively. These changes were detected 30 days after blast exposure, suggesting the possibility of long-lasting retinal injury and neuronal inflammation after primary blast exposure. The nature of 21st century military conflict has led to a dramatic increase in exposure of military personnel and civilians to blast wave pressure, leading to traumatic brain injury (TBI).1Svetlov S.I. Prima V. Glushakova O. Svetlov A. Kirk D. Gutierrez H. Serebruany V. Curley K. Wang K.K. Hayes R.L. Neuro-glial and systemic mechanisms of pathological responses in rat models of primary blast overpressure compared to "composite" blast.Front Neurol. 2012; 3: 15Crossref PubMed Scopus (75) Google Scholar The retina is part of the central nervous system; as such, it is vulnerable to injuries similar to those that affect the brain.2Choi J.H. Greene W.A. Johnson A.J. Chavko M. Cleland J.M. McCarron R.M. Wang H.C. Pathophysiology of blast-induced ocular trauma in rats after repeated exposure to low-level blast overpressure.Clin Exp Ophthalmol. 2015; 43: 239-246Crossref PubMed Scopus (34) Google Scholar Today, >80% of military personnel experiencing TBI also exhibit symptoms of visual dysfunction.3Wang H.C. Choi J.H. Greene W.A. Plamper M.L. Cortez H.E. Chavko M. Li Y. Dalle Lucca J.J. Johnson A.J. Pathophysiology of blast-induced ocular trauma with apoptosis in the retina and optic nerve.Mil Med. 2014; 179: 34-40Crossref PubMed Scopus (22) Google Scholar Primary ocular blast exposure resulting from blast wave pressure has been reported among survivors of explosions, but with limited understanding of the resulting retinal pathologies; therefore, therapeutic interventions are currently out of reach.4Zou Y.Y. Kan E.M. Lu J. Ng K.C. Tan M.H. Yao L. Ling E.A. Primary blast injury-induced lesions in the retina of adult rats.J Neuroinflammation. 2013; 10: 79Crossref PubMed Scopus (37) Google Scholar Previous consolidation of data from the US Department of Defense shows a high percentage of visual field deficits, photophobia, oculomotor dysfunction, and decrease in contrast sensitivity in service members, 45 to 60 days after blast-induced TBI, supporting the need for a chronic blast wave pressure injury model.5Magone M.T. Kwon E. Shin S.Y. Chronic visual dysfunction after blast-induced mild traumatic brain injury.J Rehabil Res Dev. 2014; 51: 71-80Crossref PubMed Scopus (50) Google Scholar Existing rodent models of blast injury show the susceptibility of the retina to the effects of low-level (120 ± 7 kPa) and high-level (≥180 kPa) blast wave pressure, including glial cell activation in the ganglion cell layer, inner nuclear layer, and outer nuclear layer, with an overall increase in biomarkers of inflammation and apoptosis.2Choi J.H. Greene W.A. Johnson A.J. Chavko M. Cleland J.M. McCarron R.M. Wang H.C. Pathophysiology of blast-induced ocular trauma in rats after repeated exposure to low-level blast overpressure.Clin Exp Ophthalmol. 2015; 43: 239-246Crossref PubMed Scopus (34) Google Scholar, 3Wang H.C. Choi J.H. Greene W.A. Plamper M.L. Cortez H.E. Chavko M. Li Y. Dalle Lucca J.J. Johnson A.J. Pathophysiology of blast-induced ocular trauma with apoptosis in the retina and optic nerve.Mil Med. 2014; 179: 34-40Crossref PubMed Scopus (22) Google Scholar, 4Zou Y.Y. Kan E.M. Lu J. Ng K.C. Tan M.H. Yao L. Ling E.A. Primary blast injury-induced lesions in the retina of adult rats.J Neuroinflammation. 2013; 10: 79Crossref PubMed Scopus (37) Google Scholar Existing models differ significantly in intensity and duration; however, long-lasting effects of blast wave pressure have not been reported. We conducted a study in which a compressed air-driven shock tube system was calibrated to deliver blast wave pressure of 300 kPa (43.5 psi) each day for 3 successive days to mice. Herein, we show that 30 days after exposure to successive blast wave pressure, the retinas of exposed mice present with glial cell activation, microglial activation, photoreceptor cell loss, and an increase in phosphorylated tau in the outer plexiform layer. Proximity of the eye to blast wave pressure had a substantial effect on the severity of these retinal responses. There was a notable difference in the response of the retina on the side of the mouse ipsilateral to blast exposure in comparison to the retina on the contralateral side. Behavioral parameters, including the Morris water maze, rotarod, and open-field activity, showed no deficits in cognitive or motor function. Neurochemical assessment of the striatum, as well as composition of astrocytes, microglia, and phosphorylated tau within the prefrontal cortex showed no indication of damage induced by blast wave pressure. To the best of our knowledge, this report is the first to compare neurological and retinal effects of blast injury. This study is also the first to show prolonged effects of blast wave pressure on specific retinal cell types. Specifically, we report Müller glia hypertrophy, microglial activation, photoreceptor cell loss, and an increase in phosphorylated tau in both retinal neurons and glial cells. This blast wave pressure model may provide insight into the underlying pathological mechanisms and help to identify markers of long-lasting retinal injury due to blast exposure. These experiments were performed in accordance with NIH's Guide for the Care and Use of Laboratory Animals6Committee for the Update of the Guide for the Care and Use of Laboratory AnimalsNational Research CouncilGuide for the Care and Use of Laboratory Animals: Eighth Edition. National Academies Press, Washington, DC2011Crossref Google Scholar and were approved by the Iowa State University Animal Care and Use Committee (protocol 4-11-7123-M). Exposure to blast wave pressure was conducted using an open-ended shock tube, as described by Shah et al.7Shah M.A. Stemper B.D. Pintar F.A. Development and characterization of an open-ended shock tube for the study of blast mtbi.Biomed Sci Instrum. 2011; 48: 393-400Google Scholar In this model, a 0.3-m driver section is rapidly pressurized with compressed helium until it ruptures four 0.35-mm-thick mylar membranes (burst pressure, approximately 9.9 MPa), propelling a shock wave down a 3-m, open-ended driven section (ID, 3.6 cm). Briefly, 20 adult male or female C57BL/6 mice were randomly divided into blast wave pressure or sham-exposed groups. Ten mice were exposed to blast wave pressure of 300 kPa (peak pressure means ± SD, 297 ± 15 kPa; positive duration, 146 ± 6 μseconds) each day for 3 successive days, and euthanized at 30 days after injury (Supplemental Table S1). The remaining 10 mice served as control. Mice were anesthetized with 4% isoflurane in 2 L/minute oxygen for 90 seconds in the induction chamber, then supplemented with 2% isoflurane in 2 L/minute oxygen via nose cone in blast animal holder until blast delivery (approximately 20 seconds). Gas anesthesia was withdrawn immediately before triggering blast. Blast winds accompanying intense blast overpressures can lead to substantial head acceleration, generating severe or lethal injuries; therefore, the animals were placed 45 degrees lateral to the shock tube axis to avoid blast wind exposure. The body was constrained securely inside a padded metal holding tube to limit blast wave pressure exposure primarily to the head. The head was constrained to limit any movement, and prevent injury due to head rotation. Control mice received anesthesia and were constrained the same way, except for exposure to blast wave pressure. Mice we placed at a distance of 15 cm from the open end of the shock tube, with the right side of the head (ipsilateral eye) facing the blast wave (Figure 1). The angle and distance from the shock tube's open end determined the peak overpressure and positive duration experienced by the mouse,7Shah M.A. Stemper B.D. Pintar F.A. Development and characterization of an open-ended shock tube for the study of blast mtbi.Biomed Sci Instrum. 2011; 48: 393-400Google Scholar both of which were measured by a pressure transducer located 1.75 cm below the mouse's head. On average, recovery from the blast exposure and anesthesia, indicated by upright posture and ambulation, occurred after approximately 30 seconds. Blast-exposed and sham mice were anesthetized i.p. with 200 mg/kg ketamine and 20 mg/kg xylazine, followed by supplementation with isoflurane, and perfused transcardially with 4% paraformaldehyde in 0.01 mol/L phosphate-buffered saline. Ipsilateral globes were post-fixed in 4% paraformaldehyde. After 24 hours, each lens was removed and globes were subjected to a sucrose gradient (10%, 20%, and 30% all in 0.1 MPO4 buffer), embedded in OCT, and frozen using dry ice. Sagittal sections (9 μm thick) of the ipsilateral retina were collected onto superfrost plus glass slides. Globes contralateral to the blast were post-fixed in Bouin's fixative for 24 hours, embedded in paraffin, and divided into sections sagitally (4 μm thick) onto superfrost plus glass slides. Paraffin-embedded sections of the retina were rehydrated using xylene, followed by a decreasing ethanol concentration gradient (100%, 90%, and 70%), and a final wash with diH2O. Heat-mediated antigen retrieval was performed using EDTA buffer (10 mmol/L Trizma base, 1 mmol/L EDTA solution, and 0.05% Tween 20, pH 9.0) in an autoclave for 30 minutes. OCT-embedded sections of the retina were hydrated for 15 minutes using tris-buffered saline with 0.05% Tween 20. Paraffin and OCT-embedded tissues were incubated with Background Buster (Innovex Biosciences Inc., Richmond, CA) for 1.5 hours. Primary antibodies against glial fibrillary acidic protein (GFAP; 1:500; Dako, Carpinteria, CA), Iba1 (1:500; Wako Chemicals USA, Inc., Richmond, VA), CD68 (1:100; Wako Chemicals USA, Inc.), AT-270 (tau pThr181; 1:100; Thermo Fisher Scientific, Inc., Rockford, IL), AT-180 (tau pThr231; 1:100; Thermo Fisher Scientific, Inc.), tau clone 39E10 (1:250; BioLegend, San Diego, CA), and calbindin (1:1000; Dako) were diluted in blocking solution containing 0.1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO), 0.04% Triton X-100 (Thermo Fisher Scientific, Inc.), and 5% normal donkey serum (Jackson ImmunoResearch, West Grove, PA) in phosphate-buffered saline and incubated for 48 hours at room temperature, then 24 hours at 4°C. Tissues were washed with tris-buffered saline with 0.05% Tween 20 (6 × 5 minutes), and incubated with a secondary antibody cocktail including Cy3 and/or Alexa Fluor 488–conjugated AffiniPure secondary antibodies (1:300; Jackson ImmunoResearch), and DAPI (1 μg/mL; Sigma-Aldrich) for 1.5 hours. After another wash, slides were mounted with Vectashield HardSet antifade mounting medium (Vector Laboratories Inc., Burlingame, CA). Negative controls were processed in parallel by omission of the primary and/or secondary antibody. The social discrimination test assesses the ability of a mouse to discriminate between its own bedding and the bedding of a mouse of the opposite sex. The test is performed inside a narrow rectangular (44.5 × 10.8 cm) chamber made of clear Plexiglas. A small container filled with bedding is fixed at either end of the chamber. Each container is sealed except for a 1-cm diameter hole on top to allow sniffing without disturbing the bedding. A 3-minute test session began by placing a mouse in the middle of the chamber facing his own bedding container. Movements inside the test chamber are video-tracked from above via webcam connected to ANY-maze software version 4.99 (ANY-maze Behavioral Tracking Software, Wood Dale, IL). The sniffing zone was defined by a circle 1 cm wider than the outside circumference of the bedding container, and time spent with head inside the sniffing zone was used to test for group differences. We assessed the impact of successive blast wave pressure exposure on learning and memory by subjecting mice to the Morris water maze, according to the rapid 2-day protocol described by Gulinello et al8Gulinello M. Gertner M. Mendoza G. Schoenfeld B.P. Oddo S. LaFerla F. Choi C.H. McBride S.M.J. Faber D.S. Validation of a 2-day water maze protocol in mice.Behav Brain Res. 2009; 196: 220-227Crossref PubMed Scopus (71) Google Scholar with some modification. The apparatus comprised a round, 1.15-m diameter galvanized stock tank filled with water mixed with white tempera paint, allowing the movements of mice to be tracked by a webcam connected to ANY-maze software. Several highly visible cues (references) were posted just outside the tank above each of four quadrants. Water temperature was maintained at 22°C to 24°C. In this 2-day rapid Morris water maze protocol, mice were subjected to 1 day of five 60-second training trials preblast wave exposure and 1 day of five 60-second training trials 30 days after exposure. During all five trials for both sessions, the 11.2-cm diameter platform (1% of pool area) was submerged 1 cm below the surface and remained in one fixed location. For trial 1 only, the platform was made visible by a bright green vertical center post marking its location. Furthermore, each mouse was placed on the platform for 10 seconds before initiating each trial. Every trial began with the mouse facing the tank wall, and trials ended with the mouse resting on the platform for 20 seconds, having either reached the platform on its own or being placed there by the experimenter after the 60-second trial ended. After each trial, mice were returned to their cages, which were placed on heating pads where they remained for 10 to 15 minutes, allowing enough time for their fur to dry before the next trial began. We assessed maze performance by measuring the time taken to first reach the platform. Locomotor coordination was measured on the accelerating rotarod (pc Rota Rod IV; Accuscan Instruments, Columbus, OH), which could test four mice per run. At 30 days after blast wave pressure exposure, mice were placed on the 3.2-cm diameter rotarod as it rotated at 0.000286 × g. Then, after a 90-second acclimation, the rotarod began accelerating to 0.007155 × g during a 1 minute (16 rpm/minute) period before ramping up to 0.007155 × g/minute for an additional 2 minutes, thus reaching a maximum speed of 0.064397 × g after 3 minutes. By accelerating to high rpm in a relatively short time, we intended this to be a demanding motor coordination task to better discriminate subtle treatment effects while simultaneously minimizing the fatigue seen in prolonged constant speed trials. Latencies to fall are reported as the average of three trials, which were executed consecutively with a 20-second gap (at 0.000286 × g) between acceleration trials. A trial ended when infrared (IR) sensors below the rod registered a fall or when the experimenter triggered the sensors manually to terminate passive rotations. In the latter case, even though the trial was terminated, mice were left on the rotarod until the trial terminated for all mice. The spontaneous locomotor activity and thigmotaxis (wall-hugging or open-field anxiety) of mice exploring a novel open field was measured in a 40 × 40-cm (1600-cm2) clear Plexiglas chamber that fit inside a VersaMax activity monitor (model VMM; Accuscan Instruments). The monitor is equipped with two 40 × 40-cm square arrays of IR beams. The IR light-emitting diodes within each array are 2.5 cm apart laterally, with the lowermost array monitoring the animal's horizontal x-y position at 1.5 cm high, whereas the uppermost array at 10.5 cm high registered vertical movements. Horizontal and vertical activity scores represent the number of IR beam breaks in the lower and upper rows of beams, respectively. The IR beam break data were acquired via the VersaMax Analyzer (model VMAUSB; Accuscan Instruments). We used VersaMap to define time spent in the center to be determined by beam breaks >10 cm (>4 IR beams) from walls. All VersaMax activity monitoring for pretreatment and post-treatment sessions lasted 12 minutes, with only the last 10 minutes used for analysis. The first 2 minutes, representing within-session acclimation, were truncated. Tissue concentrations of i) dopamine and its two main metabolites, 3,4-dihydroxyphenyl-acetic acid and homovanillic acid, ii) serotonin and its main metabolite, 5-hydroxyindoleacetic acid, and iii) norepinephrine were quantified using high-performance liquid chromatography (HPLC) with electrochemical detection. After 30 days post blast wave exposure, samples from the striatum were prepared and quantified as described previously.9Ghosh A. Saminathan H. Kanthasamy A. Anantharam V. Jin H. Sondarva G. Harischandra D.S. Qian Z. Rana A. Kanthasamy A.G. The peptidyl-prolyl isomerase Pin1 up-regulation and proapoptotic function in dopaminergic neurons: relevance to the pathogenesis of Parkinson disease.J Biol Chem. 2013; 288: 21955-21971Crossref PubMed Scopus (59) Google Scholar Briefly, four female and four male mice randomly selected from each group were sacrificed via carbon dioxide inhalation, followed by exsanguination by cardiac puncture. Target tissues dissected from the extracted brains were weighed and then immediately frozen on dry ice after suspending in 0.1 mol/L perchloric acid solution containing 0.05% Na2EDTA and 0.1% Na2S2O5 before transferring them to −80°C. Tissue homogenates were centrifuged through 0.22-μm filters before diluting 1:9 in the phosphate-buffered acetonitrile mobile phase MD-TM (ESA Inc., Chelmsford, MA). The primary analytes dopamine, serotonin, norepinephrine, and the metabolites 3,4-dihydroxyphenyl-acetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid were separated isocratically by injecting 20 μL through a C-18 reversed-phase column (Microsorb-MV 100-3; 100 × 4.6 mm; 3-μm particles) using a flow rate of 0.6 mL/minute on an HPLC system (UltiMate 3000; Dionex, Madison, WI) coupled to an analytical thermostatted autosampler (WPS-3000TSL; Dionex). The electrochemical detection system consisted of a coulometric array detector (CoulArray 5600A; Dionex) with a guard cell (model 5020; Thermo Scientific, Chelmsford, MA) and an analytical cell (model 5014B; ESA Inc.). The data acquisition and analysis were performed using CoulArray Data Station Software version 3 (ESA Inc.). Tissue homogenates were prepared using modified radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitor cocktail (Thermo Scientific, Waltham, MA), as previously described.10Harischandra D.S. Jin H. Anantharam V. Kanthasamy A. Kanthasamy A.G. α-Synuclein protects against manganese neurotoxic insult during the early stages of exposure in a dopaminergic cell model of Parkinson's disease.Toxicol Sci. 2014; 143: 454-468Crossref PubMed Scopus (73) Google Scholar Briefly, prefrontal cortex, ipsilateral to the blast was dissected and homogenized in modified radioimmunoprecipitation assay. Protein concentrations were determined with the Bradford protein assay. Homogenates containing equal amounts of protein were separated on a 10% to 15% SDS-polyacrylamide gel. After separation, proteins were transferred to a nitrocellulose membrane, and nonspecific binding sites were blocked by treating with LI-COR blocking buffer for 1 hour. The membranes were then incubated with primary antibodies directed against tau PHF-1 (pSer396,404; rabbit monoclonal; 1:2000 dilution; a gift from the laboratory of Dr. Peter Davies, Albert Einstein College of Medicine, Bronx, NY), PHF-tau clone 39E10 (mouse monoclonal; 1:2000 dilution; BioLegend, San Diego, CA), Iba1 (goat monoclonal; 1:1200 dilution; Abcam, Cambridge, MA), or GFAP (mouse monoclonal; 1:1200 dilution; Santa Cruz Biotechnology, Dallas, TX) overnight at 4°C. The primary antibody treatments were followed by treatment with IR800-conjugated anti-rabbit or Alexa Fluor 680–conjugated anti-mouse secondary antibody (1:5000; Abcam) for 1 hour at room temperature. To confirm equal protein loading, blots were reprobed with β-actin antibody (1:15,000 dilution; Abcam). Western blot images were captured with the Odyssey IR Imaging system (LI-COR Biosciences), and data were analyzed using Odyssey 2.0 software (LI-COR Biosciences, Lincoln, NE). Fluorescence images were taken at ×20 using a commercial upright microscope system (Zeiss AxioPlan 2 Microscope Imaging System; Oberkochen, Germany). Confocal images we captured using a Nikon A1R+ Resonant Scanning Confocal System with a Ti-E inverted microscope and laser lines 405, 488, 561, and 640 nm (Nikon Instruments Inc., Melville, NY). Three different sites of the retina were analyzed to gauge for differences in immunoreactivity: central retina at the thickest section of the retina adjacent to the optic nerve head, and two peripheral retinal locations on opposite sides of the retina within one 20× field of view from the peripheral margin. Micrographs were captured using a commercial photo-editing system [Adobe Photoshop and Adobe Illustrator (CC); Adobe Systems, San Jose, CA]. For quantification of GFAP, Iba1, AT-270, AT-180, and CD68 immunoreactivity, the percentage of the total image area thresholded (outer limiting membrane to inner limiting membrane of the retina) was analyzed using ImageJ version 1.47v (NIH, Bethesda, MD; http://imagej.nih.gov/ij). Sex versus treatment interaction effect via two-way analysis of variance was used to determine whether sexes should be pooled. For all behavioral as well as histological studies, the ratio of males/females was equal (n = 5 males and 5 females). Preliminary analyses for behavioral and histological studies (P ≤ 0.05) showed the two sexes did not respond differently to the same treatment (Supplemental Table S2); therefore, sexes were pooled. Pooled rotarod data were analyzed using an unpaired t-test. All other pooled behavioral data were analyzed using two-way analysis of variance, and Tukey's multiple comparisons test (post-hock). HPLC data were analyzed using an unpaired t-test. Quantified histological data were analyzed using two-way analysis of variance, and Tukey's multiple comparisons test (post-hock). Thickness of the outer nuclear layer was measured by counts of cell bodies spanning the layer. For each animal (n = 20), the central retina within a cross section was used. Retinal thickness was analyzed using an unpaired t-test. Prism 6 for Windows (Graph Pad Software, La Jolla, CA) was used for statistical analysis. Müller glia are the principal glial cells of the retina, with processes projecting radially through the thickness of the retina, and provide limits at the outer and inner limiting membrane. In the healthy retina, GFAP in Müller glia is localized to the end feet at the retina's inner limiting membrane.11Bringmann A. Pannicke T. Grosche J. Francke M. Wiedemann P. Skatchkov S.N. Osborne N.N. Reichenbach A. Müller cells in the healthy and diseased retina.Prog Retin Eye Res. 2006; 25: 397-424Crossref PubMed Scopus (1261) Google Scholar In response to retinal injury or stress, thick GFAP immunoreactive processes span the retina from the inner limiting membrane to the external limiting membrane, concomitant with hypertrophied processes of Müller glia.12Lewis G.P. Fisher S.K. Up-regulation of glial fibrillary acidic protein in response to retinal injury: its potential role in glial remodeling and a comparison to vimentin expression.Int Rev Cytol. 2003; 230: 263-290Crossref PubMed Scopus (302) Google Scholar We assessed the distribution of GFAP immunoreactivity to detect activation of Müller glia after blast exposure. GFAP immunoreactivity in sham animals with retinas ipsilateral (Figure 2A) and contralateral (Supplemental Figure S1A) to blast exposure was localized to the Müller glia end feet, and astrocytes in the optic fiber layer. When quantified, the distribution of GFAP immunoreactivity in retinas of sham animals accounted for approximately 2.60% ± 0.39% (means ± SEM) of total area (Figure 2C and Supplemental Figure S1C). Thirty days after blast exposure, the area of GFAP immunoreactivity in retinas ipsilateral to blast exposure was increased and consistently spanned the retina from the Müller glia end feet in the inner limiting membrane to the outer plexiform layer, with some apical processes visible in the outer nuclear layer (Figure 2B). There were no significant differences in the area of GFAP immunoreactivity between peripheral and central retinas of blast-exposed mice (22.79% ± 3.19% and 29.41% ± 2.35% of total area, respectively) (Figure 2C). In contrast, GFAP immunoreactivity in retinas contralateral to blast exposure remained localized to the Müller glia end feet, with occasional processes extending past the optic fiber layer (Supplemental Figure S1B). When quantified, the area of GFAP immunoreactivity in retinas contralateral to blast exposure was 4.52% ± 0.77% and 4.32% ± 0.74% of total area in peripheral and central retinas, respectively (Supplemental Figure S1C). The prevalence and morphology of retinal microglia in response to blast injury was assessed using Iba1 immunoreactivity. In sham animals, Iba1 immunoreactivity showed ramified microglia with thin irregular processes and small somata (occasionally lacking processes), within the inner and outer plexiform layers (Figure 3A). Iba1 immunoreactivity occupied 0.31% ± 0.06% of total area in peripheral retina, and 0.37% ± 0.11% of total area in central retina (Figure 3C). Iba1 immunoreactivity in the retinas ipsilateral to blast exposure showed an amoeboid-like morphology with thicker, and longer processes, and swollen cell bodies, all characteristics of activated microglia.13Karlstetter M. Scholz R. Rutar M. Wong W.T. Provis J.M. Langmann T. Retinal microglia: just bystander or target for therapy?.Prog Retin Eye Res. 2015; 45: 30-57Crossref PubMed Scopus (338) Google Scholar Iba1 immunoreactivity was localized to the inner and outer plexiform layers (Figure 3B). When quantified, Iba1 immunoreactivity accounted for 0.93% ± 0.19% and 0.93% ± 0.18% of total area in peripheral and central retinas, respectively (Figure 3C). Iba1 immunoreactivity in retinas contralateral to blast exposure was not significantly different from retinas of sham animals; however, morphological differences of microglia in retinas from blasted animals were noted (Supplemental Figure S2, A–C). The cell surface antigen CD68 is a common marker expressed on macrophages14Langmann T. Microglia activation in retinal degeneration.J Leukoc Biol. 2007; 81: 1345-1351Crossref PubMed Scopus (362) Google Scholar; therefore, immunoreactivity for CD68 was used to examine blast-exposed retinas for proinflammatory microglial activation. No CD68 immunoreactivity was detected in the retinas of sham-exposed mice (Figure 3D and Supplemental Figure S2D). The retinas of exposed mice from the side ipsilateral to the blast were positive for CD68 immunoreactivity in the inner and outer plexiform layers (Figure 3E). When quantified, CD68 immunoreactivity was 1.22% ± 0.12% and 1.16% ± 0.15% of total area in peripheral and central retinas, respectively (Figure 3F). In contrast, values for CD68-positive microglia in the retinas contralateral to blast exposure were significantly lower than those of retinas ipsilateral to blast exposure, making up 0.32% ± 0.03% and 0.30% ± 0.06% of total area in peripheral and central retinas, respectively (Supplemental Figure S2, D–F). Hyperphosphorylated tau is a major component of neurofibrillary tangles involved in the pathology of Alzheimer disease and other neurodegenerative conditions.15Lee V.M. Goedert M. Trojanowski J.Q. Neurodegenerative tauopathies.Annu Rev Neurosci. 2001; 24: 1121-1159Crossref PubM
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