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Burcu DerkuntSchool of Engineering and Applied Science, Department of Biomedical Engineering

Social ImpactDue to improved technology and augment-ed use of close-range explosives, soldiers en-counter blast brain injuries more frequently than ever. Effects of primary blast injury to the brain are the least known among its types, as well as the most difficult to diagnose. The study provides a deeper understanding of pri-mary blast brain injury by way of studying the effect of variables on the severity and location of the injury.

BiographyBurcu Derkunt, born and raised in Istanbul, Turkey, is a fourth-year Biomedical Engineering major and Engineer-ing Business minor at the University of Virginia. She decided to continue her studies in the United States at the School of Engineering Applied Sciences after finishing French high school in Istanbul. During her 3rd year at U.Va, she per-formed Brain Blast Injury research at Center for Applied Biomechanics under the guidance of Dr. Karin Rafaels. She is a member of Alpha Omega Epsilon Engineering Sorority.

Blast Brain Injury: Quantifying Axonal Injury Due to Primary Blast

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When the brain experiences disparate pressure differentials,

marked trauma occurs

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Blast Brain Injury: Quantifying Axonal Injury Due to Primary Blast

AbstractThe purpose of this experiment is to investigate the effect of pressure differentials as a source of trauma to the brain. When the brain experiences disparate pressure differentials, marked trauma occurs. These differentials can be caused by close-range explosions, whose shockwaves penetrate the cranium and damage the brain. The study of these differentials is of particular interest because increased use of close-range explosives in the Middle East poses a potential threat to American troops for developing Traumatic Brain Injury (TBI). It is hypothesized that pressure differentials in the brain incur localized damage to the diencephalon region of the cerebrum. In order to investigate the effect of pressure differentials on the brain, a live animal model is used for blast tests. Live ferrets are exposed to shockwaves of different overpressures and durations. The brains of these specimens are preserved and stained with β-amyloid precursor protein (βAPP) for axonal injury analysis. A semi-computational method, using MATLAB programming language and software, is created to quantify the axonal injury in relation to blast severity level. When the data is analyzed, it is found that an increase in the shockwaves’ pressure and duration propagate axonal injury. Regression analysis demonstrates a higher dependence on duration compared to over-pressure. Contrary to the hypothesis, damage to the brain caused by pressure differentials is diffuse, injuring axons throughout the cerebrum rather than only the diencephaplon.

Key Terms: blast injury; brain; ; primary blast; ferret; axonal injury; semi-computational model; quantification of axonal injury; traumatic blast injury

IntroductionAmong the 1.64 million military service members of US troops in Iraq and Afghanistan deployed since October 2007, approximately 320,000 have suffered from Traumatic Brain Injury (TBI) (Tanielian & Jaycox, 2008). Accord-ing to recent studies, 88% of the injuries in a medical unit in Iraq were due to improvised explosive devices, of which 47% were head injuries (Taber et al., 2006; Murray et al., 2005). Likewise, 97% of the injuries in a Marine unit in Iraq were due to explosions, of which 53% were head or neck injuries (Taber et al., 2006; Gondusky & Reiter, 2005). Al-though research has been done on blast injury, most stud-ies focused on gas-containing organs, such as lungs. The ef-fects and the impact of Blast Brain Injury are still unknown.

There are four types of blast injuries: Primary blast inju-ries caused by shockwave-induced pressure differentials; secondary blast injuries caused by fragments that are pro-pelled into the body; tertiary blast injuries caused by the body being forcefully put in motion by blast; and quater-nary blast injuries caused by any other means including burns, chemicals etc. (Knudsen & Øen, 2003; Phillips & Richmond, 1990; Stuhmiller et al., 1990). Effects of pri-mary blast injury to the brain are the least known among its types, as well as the most difficult to diagnose because it can occur without any external wounds. Hence, this study will focus on the effects of primary blast injury on the brain.

Previous studies encountered axonal injuries in animal models due to blast exposure (Saljo et al., 2000). A tech-

nique to determine the injury risk from the varying levels of blast would be to quantify the injured axons within the brain tissue and among previous studies. β-amyloid pre-cursor protein (βAPP) immunoreactivity was proven to mark damaged axons within white matter tracts (Gentle-man et al., 1993). To determine the magnitude of a blast needed to cause TBI, 25 ferrets were exposed to shock-waves of different overpressures and durations. Brains of the specimens were preserved and stained with APP for axonal injury. A semi-computational method, MATLAB code, was created to quantify the axonal injury of the blasts.

It is hypothesized that the number of axons injured will rise with the increased overpressure and increased du-ration of the shockwave; it is also predicted that dam-age incurred will be localized in the diencephalon region of the cerebrum. Effects of overpressure and duration of the blast wave will be compared. This is the first time ax-onal injury due to blast is quantified and a semi-com-putational method is used to quantify the axonal injury.

Methods Shock Tube and Test FixtureA compressed air shock tube was used to generate overpres-sure shockwaves that simulated free-field blasts. The duration was varied by either changing the length of the driver sec-tion or the high-pressure gas used, or both. The magnitude of the overpressure was determined by the number of 0.01 inch thick membranes of Mylar (DuPont Co., Wilmington, DE, USA) placed between the driver and driven sections. Three

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pressure transducers (Model 8530B, Endevco, San Juan Capistrano, CA) were located at the end of the shock tube to measure the incident (side-on) pressure of the shock tube.

Every specimen was secured into a specific rigid test fix-ture that was designed to protect the specimen’s body from the shock overpressure. Pressure inside the test fix-ture was reduced by approximately 98%, as measured by a pressure transducer inside the protective casing. A 2.0 inch thick America MatTM foam (Soundproofing Amer-ica, Inc., San Marcos, CA) and a Kevlar collar (DuPont Co., Wilmington, DE) were used to prevent neck injury. A 5.0 inch long head support extended from the fix-ture to secure the head and to prevent motion. The center of the head of the specimen was placed approximately 1.5 inches from the opening of the shock tube. The test fixture was then secured to prevent translation resulting from the blast exposure. The test fixture is illustrated in Figure 1.

Test ProcedureTwenty-three of twenty-five ferrets were tested with blasts of varying pressures and durations. Two ferrets were used as a control group. The same procedure was followed for all speci-mens, except control specimens were not exposed to blasts.

If specimens were apneic after the blast exposure, bag venti-lation was performed to bring heart rate and respiration to normal levels. In severely injured animals, either O2 or Do-pram was administered, or both. Every 15 minutes, the speci-mens’ vitals, such as heart rate, SpO2, and respiration rate, were recorded. At the end of the five-hour testing period, the specimens were euthanized with Euthasol, and a necropsy was performed. During necropsy, the heart, lungs, brain, and any other injured organs were extracted for further analysis. The extracted tissue was fixed with a 0.1 M phosphate buf-

fer solution containing 4% paraformaldehyde (Electron Mi-croscopy Sciences, Hatfield, PA, USA). After 16 hours in the fixation solution, the specimens’ brains were transferred to a 0.1 M phosphate buffer solution containing 20% sucrose. The rest of the organs were left in the fixation solution for 24 to 26 hours, and were then transferred to an ethanol solution.

The remainder of the immunohistochemical processing of the brains occurred at FD Neurotechnologies (Baltimore, MD, USA). The processing of the other organs occurred at the Biorepository and Tissue Research Facility (Charlot-tesville, VA, USA).

βAPP StainingAmyloid β-protein is derived from β-amyloid precursor pro-tein and is composed of neuritic and cerebrovascular amy-loids that are previously proven to accumulate in Alzheimer’s Disease, Down’s Syndrome, and the aging brain (Glenner & Wong, 1984). The pathophysiology of the event is still unclear, but immunohistochemically examined tests show that βAPP immunoreactivity appears within 0.5 hours after the axonal injury (Otsuka et al., 1991). In this study, extracted ferret brains were stained with βAPP in order to demonstrate axo-nal injury by using βAPP’s immunoreactivity characteristics.

Data Analysis Microscope slides of the brain tissue taken from approxi-mately 8 mm inferior to the bregma were obtained from FD Neurotechnologies and digitized using an Aperio ScanS-cope CS slide scanning system (Aperio Technologies, Inc., Vista, CA, USA) at a 40X magnification level. Follow-ing other quantitative studies of immunohistochemistry, a MATLAB (version 7.10, MathWorks, Natick, MA, USA) code was written to quantify the number of axons injured in these digitized images based on the color threshold and mor-phology differentiating the axons from the uninjured tissue (Matkowskyj et al., 2000; Rojo et al., 2009). Even though the code gave an efficacious approximation for the axonal injury area, many areas of false positive staining were still present.

An excel spreadsheet with computationally calculated axonal injury area was created such that one excel cell represented a 3000 pixel area of the digitized brain images. Each 3000 pixel area that contained positive staining was assessed man-ually for injured axons using another MATLAB code that improved the accuracy of the injured axon area by allowing for the selection or de-selection of positive area. The total in-jured area obtained from the excel spreadsheet was normal-ized with whole tissue area determined by a color threshold.

Results The specimens were analyzed according to the driver length used for blast. In this experiment, a short driver length produces a blast of higher overpressure and shorter

Figure 1. Test Fixture. The test fixture consisted of a shock tube that generated overpressure shockwaves, pressure transducers that measured the incident pressure of the shock tube, and a protective test fixture that reduced the specimen’s body exposure.

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duration, while a long driver length produces a blast of a lower overpressure and longer duration. To determine if overpressure or duration had a greater effect on axonal in-jury than the other, each factor was investigated separately.

Among specimens shot with short driver lengths, Living Fer-ret (LF) 9 and has a similar peak duration to LF 38, but LF 38 has a 23.6% higher peak pressure and a 226.5% larger axonal injury area than LF 9. The results indicate an increase in the overpressure of blast increases axonal injury by approximate-ly a factor of ten. LF 28 and LF 36 were shot with long driver lengths. LF 28 had a 27.96% higher peak duration, a 1.21% higher peak pressure, and a 2038.8% larger axonal injury area than LF 36. The results indicate an increase in the duration of blast increases axonal injury by approximately two factors of ten. These specimens prove that if the duration is aug-mented for a specimen, but the pressure remains the same, the specimen with the augmented duration would have more axonal injury than the compared specimen. As seen in Figure 2, there is a direct correlation between axon area and peak duration as well as between axon area and peak pressure.

To determine if overpressure or duration had a greater effect on axonal injury than the other, each factor was investigated separately. Although LF 35 had a 42.8% higher peak pressure, LF 28’s 10.8% higher peak duration resulted in LF 28 pro-ducing a 167.7% higher axonal injury area than LF35. When regression analysis was performed using Microsoft Excel, the long and extra-long (x-long) driver lengths gave logarithmic fits with a larger R2 value in peak duration than in peak pres-

sure in both cases (Figure 3 and Figure 4). Regression analy-ses show that peak duration has an amplified effect on axonal injury when compared to that of peak pressure. The regression analysis for short driver length was unsuccessful and a proper fit was unable to be found. The R2 value of the x-long driver length was calculated to be larger than the R2 value of long driver length in both peak duration and peak overpressure.

A graphical representation of the density of injured axons was made to illustrate the areas of axonal injury in LF 13 (Figure 5). Each square corresponds to 3,000 pixels of the undigitized cross-section of the brain. The color associated with each square correlates to a range of positive staining of injured axons on the brain and represents the intensity of the axonal injury in the specified area. The amount of positive staining and, thus, the amount of injury, increases on the color spectrum from blue to red. The results of the graphical analyses of the specimens’ brains show axonal injury due to shockwaves from close-range blasts is dif-fuse, and not localized to the diencephalon region of the cerebellum, located at the center bottom of the graphic.

Figure 2. Effects of Peak Pressure and Peak Duration on Ax-onal Injury Area. The size of the circle signifies the normalized axon area. The larger the circle is, the greater the axonal injury area. For the short driver, the duration values are analogous, and overpressure is the deterministic variable. The largest circle lies at the highest peak pressure. The plots of the long and x-long driver data show that, as the overpressure and duration increases, the size of the circle increases. This figure indicates that the circle size increases as the driver length increases. The x-long driver, with the longest duration, has the largest circles, which suggests that an increased duration increases positive axonal staining.

Figure 3. Regression Analysis for Long Driver. Regres-sion of adjusted peak pressure and adjusted peak dura-tion are shown. Logarithmic regression type is used in both graphs. The peak duration’s R2 value, 0.6738, is greater than the peak pressure’s R2 value, 0.5709, showing that the axo-nal area can be better explained with duration than pressure.

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Discussion

Random errors in axonal area between similar exposure levels may either be due to the strength and endurance of each specimen or the choice of inclusion or exclusion of various positively stained areas in calculations, or both.

Regression analyses are performed with Microsoft Ex-cel and the data used in regression curves are attached in the Appendix (Stock & Watson, 2006). Data analysis re-sulted in higher R2 values for duration than for overpres-sure, signifying that duration (ms) of a blast to the brain incurs more damage and is a better explanatory variable than overpressure (kPa) per unit. In other words, more brain damage occurs if blast duration is increased one ms more, than if the blast pressure is increased one kPa more.

Regression analyses for both long and x-long driver lengths resulted in logarithmic fits. When long and x-long driver lengths are compared, the R2 value of the x-long driver is greater than the R2 value of the long driver in both peak duration and peak pressure. For the short driver length, there was no significant regression reached; therefore, it is not in-cluded. These regression discrepancies in driver length are due to the test conditions associated with each driver length. Pressure and duration values for each driver vary. X-long driver has the widest range of duration values, long driver has a smaller range and short driver length has the smallest range. Because duration has the largest effect on the amount of in-jured axons, the shock tube configurations that have a larger range of durations can be expected to produce better fits.

It was hypothesized that the damage incurred would be lo-calized in the diencephalon region of the cerebrum, denoting either a protected region or a less endurable region of the brain, or both. If supported, this theory could result in an improved helmet with improved protection from blast brain injury. However, the results of this study show that blast in-jury in the brain has a diffuse effect with no specific area exhibiting more damage than the rest. When exposed to a blast wave the brain is exposed to volumetric and deviatoric stresses throughout the entire organ due to the differences in tissue density. These forces can cause axonal injury through-out the brain (Mendez, et al., 2005; Taber et al., 2006).

ConclusionsA semi-computational method quantifying axonal injury due to blasts provides a robust approximation of the axonal injury incurred. The MATLAB method is faster than the hand-counting method and input can be modified by the user to override suspected faulty areas of digital quantifica-tion. A direct correlation exists between injured axonal area and overpressure and duration of blasts. Regression analysis shows that duration of blasts increases axonal injury more than overpressure. The present study demonstrates that axo-nal injury caused by blast is diffuse throughout the brain.

Figure 4. Regression Analysis for X-Long driver. Re-gression of adjusted peak pressure and adjusted peak du-ration are shown. Logarithmic regression type is used in both graphs. The peak duration’s R2 value, 0.8009, is greater than the adjusted peak pressure’s R2 value, 0.7768, show-ing that the peak duration’s curve is a better fit than that of the peak pressure and may better explain the positive axo-nal area compared to pressure. R2 values for the x-long drive length are higher than the R2 values of the long driver length (Figure 3) for both peak pressure and peak duration.

Figure 5. Graphical representation of injured axon density of LF13. As the scale goes from blue to red, the amount of injury and positive staining increases. Red is the represents the most intense axonal injury and blue represents the least. Red and blue squares of different tones are dispersed throughout the brain slide. The density scale is shown on the right side of the figure, where 60 is the maximum staining and 0 is the minimum staining.

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AcknowledgementsI would like to express my gratitude to Dr. Karin Rafaels from the Department of Biomedical Engineering at the University of Virginia in Charlottesville for the opportunity to perform brain blast injury research. I deeply thank Dr. Rafaels for the expertise and supervision provided during their collaboration.

CertificationI have completed a medical health assessment as well as an online training by the Office of Animal Welfare at UVa. I successfully completed various training courses including an Orientation Seminar, Working Safely with Animals, Large Animal Surgery and Animal Facility Rules and Procedures. I am authorized to work with any species except non-human primates.

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Appendix: Test Data

Specimens were tested with 3 different driver lengths: Short, long and x-long. Charts compare the adjusted peak pressure (kPa), adjusted peak duration (ms) and the normalized axonal injury area (no units).