Hypothermia ameliorates blast-related lifespan reduction of C. elegans

Blast-related mild traumatic brain injury induces significant long-term health issues, yet treatment procedures remain underdeveloped. Therapeutic hypothermia has been postulated as a potentially effective therapy. In a Caenorhabditis elegans model, we demonstrate a dose-dependent reduction in lifespan following exposure to blast-like shock waves. Using polyvinyl alcohol, we show that cavitation is a key injurious factor in the damaging shock wave component. Short and long lifespan C. elegans mutants demonstrated the interaction of genetic and environmental longevity-determining factors. Hypothermia reduced the long term effect of shock wave exposure. Thus, we present an effective C. elegans model of long term effects of blast-related mild traumatic brain injury, as well as evidence of the merit of therapeutic hypothermia as a therapy option following blast exposure.

. Shock wave exposure decreases lifespan. (a) Lifespans of N2 C. elegans raised in liquid cultures following shock wave exposure. (b) Lifespans of N2 worms raised on NGM agar plates following shock wave exposure. (c) Lifespans of N2 worms exposed to shock waves in S-medium or polyvinyl alcohol. (d) Lifespans of N2 worms exposed to shock waves in S-medium or polyvinyl alcohol, followed by a short washing step. (e) Lifespans of daf-16(m26) worms following shock wave exposure. (f) Lifespans of N2 worms at 11 °C following shock wave exposure. (g) Lifespans of daf-2(e1370) worms at 11 °C following exposure to shock waves. In all panels, vertical dotted lines represent 50% lifespan of control. Arrows and horizontal dotted lines represent the percent survival of each test group at time of the corresponding 50% control survival. N > 500 worms in all experiment groups (the exact numbers are provided in the Methods section).
SCIeNtIFIC REPORTS | (2018) 8:10549 | DOI:10.1038/s41598-018-28910-z C. elegans to shock waves, resulted in significantly reduced immediate effect 9 . In order to investigate the role of cavitation in long term damage following shock wave exposure, we exposed C. elegans to shock waves while in PVA. Attenuating cavitation resulted in significantly longer lifespans, although still significantly lower than control-level, despite a potentially toxic effect observed when using PVA without shock waves (Fig. 1c). Any toxic effect of PVA was effectively mitigated using a short wash step prior to putting worms on NGM-agar plates (Fig. 1d).
Knockout C. elegans were previously observed with varied lifespans from wild-type N2 C. elegans. While daf-2(e1370) mutants can live more than two times longer than N2 C. elegans, the gene knockout observed in daf-16(m26) mutants also plays a role in the aging pathway 12 . In order to investigate the interaction of shock wave exposure and other lifespan shortening factors, we exposed daf-16 and daf-2 worms to shock waves. We found that exposure to shock waves induced a significant further reduction in lifespan of daf-16 worms (Fig. 1e), although the reduction in lifespan was not as pronounced as in N2 C. elegans (Fig. 1a). Following exposure to shock waves and maintenance at 11 °C, N2 ( Fig. 1f) and daf-2 worms (Fig. 1g) exhibited significant reduction of lifespan, although lifespan in daf-2 worms was still longer than that of N2 counterparts at 11 °C.
Wild-type N2 C. elegans maintained at 11 °C following shock wave exposure exhibited significantly shorter lifespan compared to control worms at 11 °C (Fig. 1f). The initial damaging effect of shock wave was evident through the first several days and similar to the N2 worms assayed at 20 °C. In the 20 °C assay (Fig. 1a), at the point when 95% of control worms remained alive (t = 8.0 days), 73.2% of worms exposed to 500 shock waves remained alive. At the 95% living mark of worms at 11 °C (t = 8.6 days) a similar amount of 74.0% of worms remained alive. However, long-term disparity between control worms and those exposed to shock waves was less evident. At the 50% survival point of control worms at 20 °C, only 9.7% and 8.2% of worms survived following exposure to 100 and 500 shock waves, respectively. For the worms assayed at 11 °C, those figures were much higher at 35.5% and 26.5%, respectively (Table 1). At the 5% survival point of worms exposed to 500 shock waves at 20 °C (t = 21.5 days), 38.3% of control worms remained alive. For worms maintained at 11 °C, only 10.4% of control worms remained alive when 5% of worms exposed to 500 shock waves remained alive (t = 27.7 days). These data imply that there is an initial death-causing effect of shock wave exposure observed in the first days, as well as a longer term effect on those worms that survive the initial damaging effect. While the initial effects do not appear to be attenuated by the lower temperature, the long-term prognosis of worms exposed to shock waves is much closer to that of control worms. The lack of improved short-term recovery in hypothermia treated worms seems to agree with previous findings of no improvement in the six month mortality rate of traumatic brain injury patients treated with hypothermia 5 . Of note, the six month recovery endpoint used in the aforementioned study correlates to approximately six hours of C. elegans recovery. Thus, the findings in the present study of attenuated long-term effects following hypothermia treatment represent findings of a different scope than in previous studies.

Conclusion
Our results show that C. elegans model the long term effects of br-mTBI by showing reduction of lifespan following exposure to shock waves. This effect is reduced in the presence of cavitation, a suspected damage causing component of primary blast waves. Furthermore, there appear to be separate short-and long-term effects of shock wave exposure on C. elegans, the latter of which can be attenuated to some extent in reduced temperature settings. This lends credence to usage of therapeutic hypothermia following br-mTBI. Given the utility of C. elegans, we believe that the model described in the present study offers a unique opportunity to further understand and investigate long term effects of br-mTBI, especially as they pertain to therapeutic approaches such as therapeutic hypothermia.

Methods
Nematodes. All C. elegans strains were obtained from the Caenorhabditis Genetics Center (Minneapolis, MN, USA), which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). The following C. elegans strains were used in the present study: Wild type (N2, Bristol), DR26 daf-16(m26), and CB1370 daf-2(e1370). From a stock liquid culture, synchronous young adult worms were produced using sodium hypochlorite treatment and sucrose cleaning as previously described 1 . Worms in liquid cultures were allowed to grow in an incubated shaker (NB 205V, N Biotek, Bucheon, South Korea) at 20 °C, while worms raised on plates were kept in an incubator at 20 °C. Assays. Seven different assays were performed in the present study. Unless otherwise noted, worms were raised in liquid cultures containing S-medium 13 and allowed to grow to the L4 stage at 20 °C. At this point, worms were cleaned using sucrose cleaning and prepared for use by concentrating via centrifugation to approximately 200 worms/mL. From this dilution, 310 µL were added to a well of a 96-well plate. When called for, shock waves were applied using a therapeutic shock wave device (Swiss DolorClast; Electro Medical Systems, S. A., Nyon, Switzerland) as previously described 9 . Following shock wave application, worms were transferred using a rapid transfer method outlined previously 9 to 10 cm NGM-agar plates containing 400 M 5-Fluoro-2′-deoxyuridine (FUdR) in order to prevent future generations of worms to hinder counting 14 . Each group contained at least 500 worms across 10 FUdR NGM-agar plates. Counting commenced on day zero, corresponding with the young adult stage, and occurred every 2 days until no living worms remained. Death was scored as the absence of any movement and failure to move at all after several light pokes with a platinum wire. The following assays were performed in the present study (the numbers in parentheses represent the numbers of worms per individual assay).
Assay 2. N2 worms were raised synchronously on 10 cm NGM-agar plates. On day zero (i.e. at the young adult stage), worms were rinsed off of plates with S-medium. Worms were centrifuged to concentrate to 200 worms/ mL. Samples were then exposed to 0, 100, or 500 shock waves (0 shock waves: 524 worms, 100 shock waves: 515 worms; 500 shock waves: 518 worms).
Data Analysis. Data was analyzed with IBM SPSS Statistics (version 23, IBM Corp., Armonk, NY) using the Kaplan Meier Mantel-Cox log rank test. A p value of 0.05 was used as the criteria for significance. GraphPad Prism (Version 5.04) was used to make Fig. 1.

Data availability.
The data that support the findings of this study are available from the corresponding author upon reasonable request.