Preservatives for postmortem brain tissue in biomechanical testing: A pilot study

Abstract Postmortem human subject (PMHS) studies are essential to brain injury research in motor vehicle safety. However, postmortem deterioration reduces the similarity between postmortem test results and in vivo response in material testing of brain tissue and in biomechanical testing of the whole head. This pilot study explores the effect of potential preservatives on brain tissue breakdown to identify promising preservatives that warrant further investigation. To identify preservatives with potential to slow postmortem degradation, samples from an initial PMHS were refrigerated at 10°C to qualitatively compare tissue breakdown from 58 to 152 h postmortem after storage in candidate solutions. On brain tissue samples from a second PMHS, compressive stiffness was measured on six samples immediately after harvest for comparison to the stiffness of 23 samples that were stored at 10°C in candidate solutions for 24 h after harvest. The candidate solutions were artificial cerebrospinal fluid (ACSF) without preservatives; ACSF with a combination of antibiotics and antifungal agents; ACSF with added sodium bicarbonate; and ACSF with both the antibiotic/antifungal combination and sodium bicarbonate. Results were analyzed using multiple linear regression of specimen stiffness on harvest lobe and storage solution to investigate potential differences in tissue stiffness. Qualitative evaluation suggested that samples stored in a solution that contained both the antibiotic/antifungal combination and sodium bicarbonate exhibited less evidence of tissue breakdown than the samples stored without preservatives or with only one of those preservatives. In compression testing, samples tested immediately after harvest were significantly stiffer than samples tested after 24 h of storage at 10°C in ACSF (difference: −0.27 N/mm, 95% confidence interval (CI): −0.50, −0.05) or ACSF with antibiotics/antifungal agents (difference: −0.32 N/mm, 95% CI: −0.59, −0.04), controlling for harvest lobe. In contrast, the stiffness of samples tested after storage in either solution containing sodium bicarbonate was not significantly different from the stiffness of samples tested at harvest. There was no significant overall difference in the mean tissue stiffness between samples from the frontal and parietal lobes, controlling for storage solution. Given the importance of PMHS studies to brain injury research, any strategy that shows promise for helping to maintain in vivo brain material properties has the potential to improve understanding of brain injury mechanisms and tolerance to head injury and warrants further investigation. These pilot study results suggest that sodium bicarbonate has the potential to reduce the deterioration of brain tissue in biomechanical testing. The results motivate further evaluation of sodium bicarbonate as a preservative for biomechanical testing using additional test subjects, more comprehensive material testing, and evaluation under a broader set of test conditions including in whole‐head testing. The effect of antibiotics and antifungal agents on brain tissue stiffness was minimal but may have been limited by the cold storage conditions in this study. Further exploration of the potential for microbial agents to preserve tissue postmortem would benefit from evaluation of the effects of storage temperature.


| INTRODUC TI ON
Testing with postmortem human subjects (PMHSs) has been used for many years to study the mechanisms of traumatic brain injuries and establish human tolerance to head injury (Depreitere et al., 2006;Nusholtz et al., 1984) and to support the development and validation of computational human body models with biofidelic head responses (Alshareef et al., 2018;Hardy et al., 2007;Nahum et al., 1977).
However, the properties of brain tissue are known to change after death.Stalnaker et al. (1977) described noticeable brain tissue degradation in whole-body PMHS tests, with excessive brain motion after periods longer than 4 days.The effects of elapsed postmortem time on the material properties of isolated brain tissue specimens have varied dramatically in previous testing.Garo et al. (2007) reported little change in porcine tissue response until 6 h postmortem and then stiffening from six to 10 h.Other studies also showed increases in stiffness over time, with Rang et al. (2001) reporting small increases in lamb brain elastic modulus from 1.5 to 14 h postmortem, McCarty et al. (2019) reporting increases in porcine instantaneous stiffness between 0 and 6.5 h postmortem, and Nicolle et al. (2004) showing a 6% increase in porcine shear modulus over 24 h.In contrast, other studies have reported softening of brain tissue.Bentil and Dupaix (2014) documented reduced stiffness in porcine brain tissue from <6 h to 1 week postmortem.Plotted results from primate tissue testing by Metz et al. (1970) showed approximately 20%-60% reductions in elastic modulus from 5 to 45 min after death.However, the authors described those results as showing little change with postmortem time compared to the larger increase in tissue stiffness that is typically produced by tissue fixation.Other studies concluded that there was little to no difference in postmortem porcine material response over 1 week (Shen et al., 2006) or with bovine tissue over three to 16 days (Darvish & Crandall, 2001).The widely varied loading modes, material properties measured, and differences in the postmortem animal or human brain tissue types tested contributed to the wide-ranging results in these studies of material properties.
Varied storage solutions and conditions would also have influenced the changes seen with elapsed postmortem time.
Potential reasons for postmortem deterioration of brain tissue include the growth of microbes (bacteria and fungi).Microbial growth is generally inhibited by colder temperatures.Therefore, slowing microbial growth may have been a factor in limiting postmortem degradation in previous studies where specimens were stored at 0-8°C prior to testing (Darvish & Crandall, 2001;Garo et al., 2007;Nicolle et al., 2004;Rang et al., 2001;Shen et al., 2006).Other methods explored previously for slowing microbial growth and associated degradation in specimens used for biomechanical testing have included high-dose radiation and modified formaldehyde storage solutions (Crandall, 1994), and antibiotics and antifungal medications (Csönge et al., 1995;Potier, 2010).Potier preserved human lower extremity tissue using a solution of amoxicillin, clavulanic acid (Ciblor®), Metronidazole (Flagyl®), and amphotericin B (Fungizone®).Csönge et al. (1995) reported that a solution containing ceftazidime, ampicillin, and amphotericin yielded better results than alternative solutions in preserving postmortem human skin.Although these studies demonstrated antibiotics and antifungals were effective in slowing degradation in other postmortem tissues, use of these methods to slow the degradation of brain tissue has not previously been reported.
Another reason for postmortem deterioration of brain tissue can be increased acidity leading to autolysis (the destruction of cells by their own enzymes).Postmortem autolysis leads to the breakdown of tissues shortly after death.In that process, cell membranes begin to break down, releasing enzymes that start digesting the cells themselves (Shedge et al., 2019).This decomposition process is triggered by increased postmortem acidity that results from a lack of oxygen and the absence of the buffering system that keeps pH balanced in vivo (Donaldson & Lamont, 2013).Clinically, elevated acidity or mean tissue stiffness between samples from the frontal and parietal lobes, controlling for storage solution.Given the importance of PMHS studies to brain injury research, any strategy that shows promise for helping to maintain in vivo brain material properties has the potential to improve understanding of brain injury mechanisms and tolerance to head injury and warrants further investigation.These pilot study results suggest that sodium bicarbonate has the potential to reduce the deterioration of brain tissue in biomechanical testing.The results motivate further evaluation of sodium bicarbonate as a preservative for biomechanical testing using additional test subjects, more comprehensive material testing, and evaluation under a broader set of test conditions including in whole-head testing.The effect of antibiotics and antifungal agents on brain tissue stiffness was minimal but may have been limited by the cold storage conditions in this study.
Further exploration of the potential for microbial agents to preserve tissue postmortem would benefit from evaluation of the effects of storage temperature.
antibiotics, antimicrobial, biomechanics, brain tissue stiffness, sodium bicarbonate, storage temperature, tissue preservatives acidosis in living patients has been treated with sodium bicarbonate (NaHCO 3 ), which acts as a buffer to increase blood pH and decrease acidity (Fujii et al., 2019).Sodium bicarbonate has also been used as a buffer in embalming solutions to preserve cadavers for dissection (Brenner, 2014).Use of sodium bicarbonate to reduce tissue degradation associated with postmortem acidity has not been reported previously in postmortem biomechanics testing, but sodium bicarbonate's ability to reduce acidity suggests that it has the potential to reduce or slow postmortem autolysis and cell membrane breakdown.
The development of procedures for reducing postmortem degradation of brain tissue could improve the biofidelity of the postmortem brain as a model for the material properties of in vivo brain tissue and the mechanical response of the whole brain in trauma testing.Additionally, slowing the processes that lead to a breakdown of brain tissue could extend the time after death during which testing of postmortem brain tissue could be performed.Therefore, the objective of this pilot study was to explore the potential for solutions containing antibiotic and antifungal agents and/or sodium bicarbonate to slow postmortem breakdown of human brain tissue in order to identify promising preservatives for further investigation.

| Qualitative evaluation
Brain tissue samples from the first of two subjects obtained through the body donor program at The Ohio State University were harvested at 58 h postmortem for qualitative assessment of tissue integrity after storage in candidate preservative solutions.The subject was male and 67 years old.
To harvest the brain tissue samples, the skull vault was removed with a surgical craniotome and an autopsy saw.The dura was opened to expose the brain, which was then removed from the skull.Brain tissue samples were obtained from the same frontal lobe to reduce the potential for harvest location to confound the results.Four samples of approximately 25 mm depth were taken from the surface of the brain using a surgical scalpel, cutting perpendicular to the brain surface.Samples included gray and white matter tissue.
After harvest, specimens were stored in one of the following artificial cerebrospinal fluid (ACSF) solutions, with or without preservatives: • ACSF only: artificial cerebrospinal fluid was prepared with distilled water using the recipe of salts, acid salts, buffering components, and glucose published by Sugawara et al. (1996).Because the ACSF is isotonic to physiological CSF, it was expected to minimize the diffusion of fluid into or out of the tissue.No preservatives were added.
• ACSF+ABX/AF + NaHCO 3 : ACSF with the same concentration of antibiotics and antifungal medications as in the ACSF+ABX/ AF solution (500 mg ceftazidime, 1000 mg ampicillin, and 80 mg amphotericin per liter) as well as the same concentration of added sodium bicarbonate that was in the ACSF+NaHCO 3 solution (3 g NaHCO 3 per liter).
Four samples, one stored in each storage solution, were kept refrigerated at 10°C until 152 h postmortem for qualitative evaluation of tissue breakdown.The solutions were not refreshed during that time.The appearance of the specimens stored in each solution was compared at 58, 83, 109, and 152 h postmortem.

| Compression testing
A second PMHS was obtained for compression testing of brain tissue to further evaluate preservative solutions that were identified in the qualitative evaluation as having potential to reduce tissue degradation.The subject was male and 80 years old.Brain samples were harvested from the parietal (n = 16) and frontal lobes (n = 14) between 32 and 35 h postmortem with a 16 mm diameter cylindrical die (Figure 1).For each sample, the cutting die was positioned perpendicular to the surface of the brain and pushed through the brain so that the cylindrical tissue sample could be retrieved from the other side.Each sample was trimmed to 25 mm in length, retaining the cortical surface at the top of the sample so that each sample was from tissue in the most superficial 25 mm of the brain.

| Qualitative evaluation
In the qualitative analysis, all specimens remained intact at 83 h postmortem (25 h after harvest) but appeared slightly swollen compared to their condition at harvest (Figure 3).The storage solutions that  samples (11 frontal and 13 parietal) were stored at 10°C for approximately 24 h after harvest before undergoing compression testing.The samples that were tested 24 h after harvest were stored in artificial cerebrospinal fluid (ACSF), or ACSF solutions with antibiotic and antifungal medications (ACSF+ABX/AF), sodium bicarbonate (ACSF+NaHCO3), or a combination of both (ACSF+ABX/ AF + NaHCO3).Compression test results are summarized in Table 1 and Table S1 (see the Online Supplement).A sample force-displacement plot is included in the Online Supplement (Figure S1).

| Compression testing
The frontal sample stored at 10°C for 24 h in ACSF+ABX/ AF + NaHCO3 with estimated stiffness of 1.82 N/mm was stiffer than any other sample by more than 0.7 N/mm (Table 1) and was determined to be influential.Given that its calculated stiffness was unrealistically higher than other samples (four standard deviations above the mean stiffness of all samples, regardless of lobe or storage solution), it was excluded from the regression analysis reported below.Results with this outlier included can be found in the Online Supplement (Table S2).
No significant difference in specimen stiffness was observed between specimens harvested from the frontal and parietal lobes, controlling for storage conditions using linear regression (Table 2).
Adjusting for the effects of storage solution, samples harvested from the frontal lobe had an adjusted mean stiffness of 0.52 N/mm compared to the 0.54 N/mm observed in samples harvested from the parietal lobe.The adjusted mean stiffness of samples tested immediately

TA B L E 2
The association between specimen stiffness, harvest lobe, and solution type.b Adjusting for harvest lobe.
after harvest was 0.70 N/mm (95% CI: 0.54, 0.86).Samples stored in ACSF at 10°C for 24 h were 0.27 N/mm (95% CI: −0.50, −0.05) less stiff than the samples tested at harvest when controlling for lobe and that difference was statistically significant.Similarly, samples stored in ACSF+ABX/AF at 10°C for 24 h were significantly softer than samples at harvest when controlling for lobe.The adjusted mean stiffnesses of samples stored in sodium bicarbonate (ACSF+NaH CO 3 : 0.60 N/mm and ACSF+ABX/AF + NaHCO 3 : 0.52 N/mm) were lower than the adjusted mean stiffness of samples tested at harvest (0.70 N/mm) though these reductions were not statistically significant.

| DISCUSS ION
In biomechanics research, delaying postmortem tissue deterioration could extend the time available for PMHS testing before the brain tissue deteriorates too much to be usable for material testing, Potential contributors to postmortem tissue degradation include microbial growth and the destruction or autolysis of cells as a result of elevated acidity.Antibiotics and antifungal medications have previously been found to be successful at slowing degradation in other body regions (Csönge et al., 1995;Potier, 2010) but are not known to have been tested on brain tissue.The authors are unaware of any previous use of sodium bicarbonate as a preservative for postmortem biomechanics research involving any body part.
The apparent success of cool storage temperatures in reducing postmortem changes in brain tissue material properties in previous testing (Darvish & Crandall, 2001;Garo et al., 2007;Nicolle et al., 2004;Rang et al., 2001;Shen et al., 2006) may be linked to the inhibition of microbial growth at lower temperatures.Comparisons of porcine brain material properties after storage at temperatures ranging from ice cold to body temperature (Rashid et al., 2013;Zhang et al., 2011)  Accordingly, all of the tissue samples in the current study, aside from the specimens tested at harvest, were refrigerated between harvest and testing.The 10°C refrigeration temperature was warmer than in many previously reported specimen tests but closer to the temperatures that could be consistently maintained in whole-head testing.
As a result, this study of the incremental benefit of storing the brain tissue in candidate preservative solutions is specifically relevant for tissue stored and/or tested in cool conditions.
Qualitative evaluation of specimens from the first subject at 83 h postmortem (after 25 h of storage) showed that specimens stored in ACSF with added sodium bicarbonate, with or without antibiotics and antifungal medications, maintained their shape better than those stored in ACSF solutions without added sodium bicarbonate.Solutions containing added sodium bicarbonate also remained clearer than solutions without added sodium bicarbonate.These qualitative findings suggested that less tissue had disintegrated or dispersed into the solutions containing sodium bicarbonate, a promising sign that sodium bicarbonate may be effective as a tissue preservative over these time ranges.Over longer time ranges, up to 94 h of storage (152 h postmortem), the sodium bicarbonate solution that also contained antibiotics and antifungal ingredients was clearer than the sodium bicarbonate solution without antibiotics and antifungal medications, suggesting that antimicrobials may also show promise as brain tissue preservatives.This preliminary qualitative assessment supported the inclusion of sodium bicarbonate and antimicrobial solutions, separately and combined, in the subsequent experimental compression testing.
Using a free-falling mass to test dynamic compressive stiffness of the tissue samples was the simplest available approach for characterizing tissue changes relevant to injury biomechanics testing.
Overall, the pilot compression tests indicated a drop in tissue stiffness, that is, softening, between the time of harvest and after 24 h of cold storage.For samples stored in ACSF without preservatives or in ACSF with antibiotics and antifungal agents, the adjusted mean drop in stiffness was statistically significant so that specimens were substantially softer after 24 h of storage.However, for samples stored in solutions that contained sodium bicarbonate, with or without antibiotics and antifungal agents, the reduction in stiffness within 24 h of cold storage was statistically insignificant.This result suggests that solutions containing sodium bicarbonate show particular promise for slowing postmortem softening of brain tissue in these time and temperature ranges.Sodium bicarbonate's potential for slowing postmortem degradation of brain tissue could be related to its buffering properties, which can neutralize the postmortem increase in acidity that triggers autolysis to break down cells.The more limited effectiveness of antibiotics and antifungal components for preserving tissue stiffness may have resulted from the cooler temperatures used for storage in this test series.It has been reported that antimicrobial agents are more effective at higher temperatures, with kill rates increasing at a ratio between 1 and 1.5 per 1°C increase in temperature for most disinfectants and preservatives (Russell, 2004).The drop in effectiveness with cooler temperatures has been confirmed experimentally for several antimicrobial agents, including antibiotics (Alonso-Hernando et al., 2013;Hajdu et al., 2010;Hinks et al., 1977).
Sample storage at 10°C in this test series may have limited the effectiveness of the antibiotics and antifungal solutions in the current study, in contrast to previous studies that showed their success in preserving other tissues.As a result, any findings related to the antibacterial and antifungal solutions in the current study only apply at the storage temperature used in this pilot study.Based on the results from the qualitative comparison of tissue stored in candidate solutions, it is also possible that the effectiveness of antibacterial and antifungal solutions may be greater over longer storage times than those used in the experimental compression testing study.
Ultimately, more comprehensive testing is needed to draw conclusions about the effectiveness of candidate preservatives.An increased sample size is needed to confirm the promising results associated with sodium bicarbonate in this study, and more comprehensive testing under test conditions beyond those used in this pilot study would be needed to generalize the results to other test scenarios.Specifically, in addition to changes in the compressive stiffness that could be measured using the very simple free-falling mass approach reported in this study, shear and viscoelastic properties of the brain would be needed to understand the effects of preservation solutions in loading conditions relevant to biomechanical testing.Linear force deflections were observed in this study.
However, it should be noted that the brain tissue is a viscoelastic material.The elastic behavior of the brain response from the current study is likely a result of the combination of the small dropped mass and the testing mode.Unlike a material testing machine that applies a constant loading rate, it is difficult to achieve a constant loading rate with a free-fall mass since the mass is decelerated as the brain tissue is compressed.Although this this simple compressive test procedure provided sufficient information about the effect of storage solutions on brain response to identify potential preservatives for future exploration, the deceleration during loading is a limitation of the testing setup.Additionally, testing with a wide range of postmortem harvest times, storage durations, storage temperatures, sampling locations in the brain, and tissue sample dimensions would be required to more thoroughly evaluate the potential effects of preservatives on brain tissue response in biomechanical testing.
Further investigation would also be needed to assess the applicability of results to whole-head testing.Although the brain may deteriorate more slowly in intact-head testing than in specimen testing that exposes the tissue to air, the use of preservatives may be more challenging in whole-head testing since tissue cannot be submerged in fluid, and preservative fluids must be perfused in the vasculature or cerebrospinal fluid compartments.

| CON CLUS IONS
The results of this study (1) introduce sodium bicarbonate as a po-

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

R E FE R E N C E S
Alonso-Hernando, A., Guevara-Franco, J.A., Alonso-Calleja, C. & Capita, R. ( 2013) Effect of the temperature of the dipping solution on the antimicrobial effectiveness of various chemical decontaminants After harvest, each specimen was either placed briefly into the ACSF before immediate compression testing (n = 3 parietal; n = 3 frontal) or stored at 10°C for 24 h prior to compression testing (n = 13 parietal; n = 11 frontal).Each specimen was tested only once.Parietal specimens were stored in one of the following four solutions: ACSF only, ACSF+ABX/AF, ACSF+NaHCO 3 , or ACSF+ABX/AF + NaHCO 3 .Frontal specimens were stored in either F I G U R E 1 Harvest of frontal lobe test specimen with cylindrical cutting die.ACSF only or the ACSF solution with all candidate preservatives (ACSF+ABX/AF + NaHCO 3 ).Compression testing was performed with a free-falling mass.Each sample was placed in a custom-designed compression testing fixture with the cortical surface facing up.A glass tube was positioned above the sample to guide the 64 mm free-fall of the 20-g mass (Figure2).The glass drop tube was positioned vertically in the fixture for each sample so that the drop tube supported only the top edge of the sample.This configuration kept the sample upright prior to testing but left it unconstrained to allow outward expansion of the sample during compression.Ambient temperature during testing was room temperature (nominally 20°C).Force and displacement data were recorded for each test.Compression forces were measured with a 22.7 kg (50 lb) capacity load cell [Transducer Techniques, Temecula, California, USA] positioned under the compression fixture.Force data at the load cell were collected at 20,000 Hz using a SLICEPRO data acquisition system [DTS, Seal Beach, California, USA].Force-time results were filtered at Channel Filter Class (CFC) 180 (SAE, 2007).Compression of the sample during testing was estimated by tracking the displacement of the free-falling mass captured using a high-speed video camera [AMETEK MiroM320S, Simi Valley, California, USA] that was positioned to be centered at the top of the specimen.The camera used a 50 mm lens, and video was collected at 8000 Hz.Displacement of the mass from initial contact with the specimen to the point of maximum sample deformation was tracked from the video images using TEMA motion analysis software (TEMA Version 3.7, Image Systems, Linköping, Sweden).Stiffness of each sample was estimated using a linear fit of the slope of the force-displacement curve in the range of 20%-80% of the peak force.Stiffness data were tabulated by harvest lobe and storage condition (time and storage solution).Mean stiffness and standard error were calculated for each lobe/storage grouping.Multiple linear regression was used to investigate differences in sample stiffness across both the harvest lobe and storage condition (Equation1).Harvest lobe was included in the model as a two-level categorical variable: frontal lobe (reference) and parietal lobe because of potential regional differences in brain mechanical properties(Prange et al., 2000;Takamura et al., 2020).Storage condition was included as a five-level categorical variable: harvest (tested immediately without storage, reference) and the following solutions where the specimen was tested after 24 h of storage at 10°C: ACSF, ACSF+ABX/AF, ACSF+NaHCO3, and ACSF+ABX/AF + NaHCO3.Each specimen was tested only once.where β 0 is the mean stiffness of frontal specimens tested at harvest; parietal is a categorical variable set to 1 for parietal specimens; ACSF, ACSF + ABX, ACSF + NaHCO 3 , and ACSF+ ABX/AF + NaHCO3 are binary indicator variables corresponding to storage solution; and β 1 , β 2 , β 3 , β 4 , and β 5 are regression coefficients representing the mean difference that each variable makes relative to stiffness.After the initial regression analysis, studentized residual, leverage, Cook's D, and DF (degrees of freedom) beta plots were used to identify potential outliers.Analyses were run with and without identified outliers.All analyses were conducted using Stata 15.1 (StataCorp LLC, College Station, TX, USA) with statistical significance set at an alpha level of 0.05.
contained sodium bicarbonate (ACSF+NaHCO 3 and ACSF+ABX/ AF + NaHCO3) were clearer than ACSF and ACSF+ABX/AF storage solutions up to 83 h postmortem.At 109 h postmortem (51 h after harvest), ACSF+ABX/AF + NaHCO3 was more transparent than the other solutions.By 152 h postmortem (94 h after harvest), ACSF+ABX/AF + NaHCO3 still remained clear.The results suggested that samples stored at 10°C in the solutions that contained sodium bicarbonate showed less evidence of tissue breakdown into the storage solution and maintained the tissue shape effectively at 83 h postmortem after 25 h of storage.At 152 h postmortem, after 94 h of storage at 10°C, the samples stored in ACSF solution with both antibiotic/antifungal medications and sodium bicarbonate (ACSF+ABX/AF + NaHCO3) showed less evidence of tissue breakdown into the storage solution than the test samples stored without preservatives or with only one of those candidate preservatives.

F
Specimen appearance in storage solution from time of harvest (58 h postmortem) to 152 h postmortem.TA B L E 1 Stiffness by lobe and storage solution.
kinematic tracking, or injury tolerance testing.Reduction of postmortem degradation of brain tissue also has the potential to improve the reliability of results from postmortem tissue testing and whole-body PMHS testing investigating brain injuries.The current pilot study used qualitative assessment and compression testing on isolated samples of brain tissue to compare changes in integrity and stiffness of samples evaluated at harvest and after cold storage in solutions containing the candidate preservatives.With only one subject each for qualitative assessment and compression testing, the results are neither definitive nor comprehensive.However, since the antibiotic/antifungal solutions used in this study had not previously been used to preserve postmortem brain tissue in biomechanics testing and sodium bicarbonate had never been used to preserve any tissue in PMHS biomechanical testing, these pilot results provide crucial insight and direction for further investigation.
may further support the effectiveness of cooler temperatures for slowing postmortem degradation.Both studies concluded that colder storage temperatures inhibited tissue degradation over the postmortem timeframes investigated, although these lower temperatures resulted in stiffer responses in Rashid's tests and softer responses in Zhang's tests.However, while these previous studies support that cool storage temperatures may effectively slow postmortem degradation, it is not expected that they could be relied on to arrest it over the days-long periods needed to prepare and test PMHS heads for biomechanical testing.Additionally, controlling the temperature of postmortem human subject heads between the time of death and testing is challenging: Researchers may have little control over storage temperature prior to subject preparation and testing and maintaining the brain at consistently low temperatures without freezing the tissue is practically challenging in whole-head testing.Therefore, cooling PMHS heads and brain tissue prior to biomechanical testing is likely good practice, but additional methods are needed to slow degradation resulting from microbial growth.
tential new tool for reducing the deterioration of brain tissue in biomechanical testing and (2) provide preliminary insights into the limited effect of antibiotic and antifungal solutions at cooler temperatures for preserving brain tissue.Given the importance of postmortem testing for studying the biomechanics of brain injury, any improvement in methods for maintaining in vivo-like brain response has the potential to improve understanding of brain injury mechanism and tolerance to head injury.The results of this study motivate further evaluation of sodium bicarbonate as a tissue preservative for biomechanical testing.The combined effects of storage temperature and storage time with different antimicrobial compounds also warrant additional evaluation.AUTH O R CO NTR I B UTI O N S Experimental concept and design: AW, AM, YK, HR, and KM.Identification of candidate preservatives: AW, AM, and YK.Literature review: AW, AM, and AT.Qualitative study data collection: AW and YK.Quantitative study data collection: AW, YK, CT, AM, and HR.Statistical analysis: LN, KS, and AM.Manuscript preparation: AM, AW, YK, TA, and LN.Critical review of manuscript: HR and KM.ACK N OWLED G M ENTS First, we are deeply grateful to the anatomical donors.Without these selfless gifts, it would not be possible to conduct this research.The authors also thank Bernie Cook (The Ohio State University and University of Minnesota) for his contributions to the preparation and execution of these experiments, as well as Tyler Arquette and Scott Bazzle (Transportation Research Center Inc.) for preparation of the manuscript and figures.

Lobe Storage time (hours) Storage solution Number of specimens (n) Stiffness (N/mm) Mean stiffness (SE) (N/mm)
Note: Mean stiffness (SE) including the potential outlier: 0.71 (0.23) N/mm. a Identified as a potential outlier.bExcluding the potential outlier.