Oral ascorbic acid 2-glucoside prevents coordination disorder induced via laser-induced shock waves in rat brain.

Oxidative stress is considered to be involved in the pathogenesis of primary blast-related traumatic brain injury (bTBI). We evaluated the effects of ascorbic acid 2-glucoside (AA2G), a well-known antioxidant, to control oxidative stress in rat brain exposed to laser-induced shock waves (LISWs). The design consisted of a controlled animal study using male 10-week-old Sprague-Dawley rats. The study was conducted at the University research laboratory. Low-impulse (54 Pa•s) LISWs were transcranially applied to rat brain. Rats were randomized to control group (anesthesia and head shaving, n = 10), LISW group (anesthesia, head shaving and LISW application, n = 10) or LISW + post AA2G group (AA2G administration after LISW application, n = 10) in the first study. In another study, rats were randomized to control group (n = 10), LISW group (n = 10) or LISW + pre and post AA2G group (AA2G administration before and after LISW application, n = 10). The measured outcomes were as follows: (i) motor function assessed by accelerating rotarod test; (ii) levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), an oxidative stress marker; (iii) ascorbic acid in each group of rats. Ascorbic acid levels were significantly decreased and 8-OHdG levels were significantly increased in the cerebellum of the LISW group. Motor coordination disorder was also observed in the group. Prophylactic AA2G administration significantly increased the ascorbic acid levels, reduced oxidative stress and mitigated the motor dysfunction. In contrast, the effects of therapeutic AA2G administration alone were limited. The results suggest that the prophylactic administration of ascorbic acid can reduce shock wave-related oxidative stress and prevented motor dysfunction in rats.

Introduction

Blast injury has been a constant threat in recent conflicts because of frequent terrorist attacks using weapons such as improvised explosive devices (IEDs). These changes of combat style affect both civilian and military populations and result in a large number of patients suffering from blast-related traumatic brain injury (bTBI) [1-3].

Notably, most bTBI patients who are exposed to low or mild blast exposure lack any external physical evidences and abnormalities detected by conventional imaging devices; therefore, they are being diagnosed as having blast-related mild TBI (bmTBI). However, during the chronic phase, they develop persistent physiological and psychological changes associated with higher-order brain dysfunction during the chronic phase [4, 5]. The precise mechanisms of such bmTBI are complicated; however, studies have shown that blood-brain barrier (BBB) disruption [6], brain edema [7], and neuroinflammation [8] play certain roles in the development of bmTBI. It was also reported that oxidative stress was involved in the pathogenesis of bmTBI [9, 10] and that an antioxidant ameliorated behavioral deficits in a bmTBI model [11]. Oxidative stress has been implicated in multiple models of TBI [12, 13] and is mainly induced by reactive oxidative species (ROS) such as superoxide, hydroxyl radical, and hydrogen peroxide [14, 15]. While basal levels of ROS are present during normal redox reactions and the electron transport chain, excess amounts, as seen after injury, can be harmful. NADPH oxidase (NOX) is a multi-subunit enzyme that catalyzes the formation of superoxide radicals from available molecular oxygen. NOX is upregulated in multiple brain regions following blast injury, and neurons maximally contributes to a higher increase in the hippocampus compared with other neural cells [16, 17]. Moreover, oxidative stress also contributes to enhanced BBB permeability during bTBI via a pathway that involves increased matrix metalloproteinase activation [18].

Hydrogen sulfide, which is one of the antioxidants, also affected the controlled cortical impact injury in rats, leading to improved neurologic dysfunction, increased activities of endogenous antioxidant enzymes (superoxide dismutase and catalase), decreased levels of oxidative products (malondialdehyde and 8-iso-prostaglandin F2α), increased BBB permeability, and attenuation of brain edema. Furthermore, the use of the KATP channel blocker, 5-hydroxydecanoate, further demonstrated activation of mitochondrial adenosine triphosphate sensitive potassium channels, and oxidative stress was reduced following treatment with exogenous hydrogen sulfide [19, 20].

To investigate blast injuries, we used laser-induced shock waves (LISWs), which have advantages including safety, ease to use, compact device, versatility, and highly controllable shock wave energy [21], as compared with other shock wave sources such as micro actual explosions [22] and shock [23, 24]/blast [25] tubes. It is believed that the above-described bmTBI symptoms are associated with the primary mechanism, that is, the effects of the blast shock wave. Since LISWs are pure shock waves with no dynamic pressure component, LISWs could represent a useful tool to investigate the primary mechanisms of blast injuries.

In this study, considering the ability to exert strong antioxidant activity by acting as free radical scavenger [26-29], we evaluated the effects of ascorbic acid 2-glucoside (AA2G), a stable derivative of ascorbic acid which is generated by binding glucose to conventional ascorbic acid [30], on the LISW-induced TBI model.

Materials and methods

Animals

Male 10-week-old Sprague-Dawley rats, weighing 310 to 380 g, were obtained from Japan SLC, Inc. (Shizuoka, Japan) and were provided with water and standard chow ad libitum. They housed two per cage and maintained at a constant temperature (21o – 23°C) and humidity (40–60%) with lights on 06:00–18:00. They were housed for 1 week in their home cages with a 12-h light-dark cycle before beginning any procedures. The rats were anesthetized with isoflurane (induction: 5%, maintenance: 2%) and their head were shaved to apply LISWs through intact scalp before the LISW application. All animals were closely monitored post-LISW application with weight and health surveillance recording as per IACUC guidelines. After the assessment of motor function, rats were perfused with saline and were sacrificed with decapitation by an experienced animal technician under deep anesthesia by intraperitoneal injection of 100 mg/kg pentobarbital sodium, and their brains were removed and were cut into cerebrum, hippocampus and cerebellum tissues. All animal experiments were conducted in accordance with the National Defense Medical College guidelines for the care and use of laboratory animals in research. The study protocol was approved by the Animal Ethics Committee of the National Defense Medical College (#16036).

Administration of ascorbic acid

AA2G (Hayashibara Co., Okayama, Japan) was dissolved in distilled drinking water and was administered daily per os (p.o.) to rats at a dose of 250 mg/kg/day (250 mg of AA2G contained 125 mg/kg/day of ascorbic acid) after LISW application for a total of 7 days (LISW + post AA2G group). LISW group rats in this experiment also received p.o. pure distilled water after LISW application for 7 days (Fig 1A). Control group also received p.o. pure distilled water for the same period.

Fig 1

The experimental protocol illustrating application of LISW and administration of AA2G.

(A) The experimental design showing therapeutic administration of ascorbic acid 2-glucoside (post AA2G). Rats were exposed to laser-induced shock wave (LISW) or anesthesia, and their motor function was assessed using a rotarod apparatus 3 day before and 7 days after the procedures. AA2G (250 mg/kg/day) or distilled water was administered only after the procedures for a total of 7 days. Male 10-week-old Sprague-Dawley rats were used (n = 10 in each experimental group). (B) The experimental protocol illustrating prophylactic and therapeutic administration of AA2G (pre and post AA2G). In this experiment, motor functions were assessed 3 days before and 7 days after the procedures. AA2G or distilled water was administered from 3 days before the procedures for a total of 10 days. Male 10-week-old Sprague-Dawley rats were used (n = 10 in each experimental group).

In another experiment, AA2G was administered daily to rats from 3 days before LISW application to 7 days after the LISW for a total of 10 days (LISW + pre and post AA2G group). Control and LISW groups received p.o. pure distilled water for the same period (Fig 1B).

Generation and characteristics of LISW

The method for generation of an LISW is shown in Fig 2A. A laser target, which is a laser-absorbing material (0.5-mm-thick natural black rubber disk) on which an optically transparent material [1.0-mm-thick polyethyleneterephthalate (PET) sheet] is adhered, was placed on the tissue. The target was irradiated with a short laser pulse, which is absorbed by the rubber to induce a plasma, and its expansion is accompanied by a shock wave (LISW). This was a type of microexplosion process from a physics point of view. In the present study, the second harmonics of a Q-switched Nd:YAG laser (Brilliant b, Quantel, Les Ulis Cedex, France; wavelength, 532 nm; pulse width, 6 ns) was used, and the scheme was the same as those used for our previous studies [31-36]. Fig 2B shows typical temporal pressure profiles of LISWs generated at different laser fluences on the target. No pressure signals were detected beyond the time range (after 2 μs; Fig 2B). Shock wave energy of LISWs is highly controllable; the peak pressure increases monotonically with increasing laser fluence (Fig 2C) and the size of the wave source can be changed by changing the laser spot size on the target.

Fig 2

Generation and characteristics of laser-induced shock waves (LISWs).

(A) Setup for generating a LISW. (B) Typical temporal waveforms of LISWs at different laser fluences on the laser target. (C) Dependence of peak pressure of LISW on laser fluence.

No dynamic pressure is produced in the generation of LISWs; therefore, the effects of acceleration or displacement can be excluded in animal studies, enabling analysis of the primary mechanism of bTBI. Positive pressure duration (hereafter simply termed duration) is an important parameter to examine shock wave to brain interactions. The duration of a typical IED explosion-related shock wave ranges from 200 μs to several milliseconds [37, 38]; however, the interaction of the human brain with such IED-related shock waves, especially brain to skull boundary effects, cannot be reproduced in the brains of small animals using when similar duration shock waves. Therefore, a scaling law that considers the anatomical differences between human brain and animal brains should be used. However, few studies have focused on this important issue. In the present study, we assumed that the impulse (time-integrated positive pressure component) of the shock wave was the primary parameter for determining brain injury. Alley previously estimated impulses of various types of IEDs as a function of propagation distance ranging from ~6.9 to ~100 Pa•s for propagation distances of 1–10 m [37]. Although the duration of LISWs is approximately two to three orders of magnitude shorter than that of typical IED shock waves, their impulses can be easily controlled by changing the laser fluence and hence the peak pressure of LISW. IED impulses within this range can be replicated with LISWs. The limitations associated with this assumption are described in the Discussion and Limitations section.

LISW application

The rats were anesthetized with isoflurane (induction: 5%, maintenance: 2%) and their head were shaved to apply LISWs through intact scalp. A laser target (a rubber disk of 8 mm in diameter covered with a PET sheet) was held with forceps and placed on the head skin of the rats; ultrasound gel (Hitachi Aloka Medical, Tokyo, Japan) was used between the bottom of the rubber and the head skin for acoustic impedance matching. For each application, rats were subjected to a set of four LISW pulses on the following sites of the scalp to cover the whole brain: 5 mm bilateral from a point at the intersection of auricular line with mid-sagittal line and then 5 mm anterior from each point. The laser spot size and fluence on the target were kept constant at 3 mm and 2.4 J/cm2, respectively, producing an LISW with an impulse of 54 Pa•s. Mishra et al. examined physiological and pathological changes, as well as changes in blood-borne biomarkers in rats exposed to blasts with a wide range of overpressure and impulses using a shock tube [17]. They determined the conditions under which mild, moderate, severe, and lethal bTBIs were induced, and postulated that mild bTBI was caused by blasts with overpressures ≤145 kPa, with a corresponding impulse of 250 Pa•s. The impulse used in this study (54 Pa•s) was within this range. The control rats were subjected to the same procedures without receiving LISW (sham injury).

Assessment of motor function

Before and seven days after LISW application or sham injury, rats were tested using a rotarod apparatus (Penlab, Barcelona, Spain), by which abnormalities in pathways responsible for integrated vestibulomotor and sensorimotor function can be evaluated [39]. Motor function of the rats was assessed by measuring the latency (in second) during which the rats remained on a 60-mm-diameter rod rotating with an initial velocity of 4 rpm and an acceleration of 8 rpm. After one acclimatization session on the rotarod apparatus, rats received five trials at 5-minute intervals. Each trial was terminated when the rat fell completely off the rod or gripped the rod and rotated in a (spun around one) complete revolution and time on the rod was recorded. The trials with shortest and longest time on the rod were eliminated and the average time on the rod of the three remaining trials was used for statistical analyses, according to previously published studies [40, 41].

Protein extraction

After the assessment of motor function, rats were perfused with saline and were sacrificed by decapitation under deep anesthesia by intraperitoneal injection of 100 mg/kg pentobarbital sodium, and their brains were removed and were cut into cerebrum, hippocampus and cerebellum tissues. At this point, it was observed that none of rats used showed apparent gross hemorrhage. Protein samples of the obtained brain tissues were extracted in a cell lysis buffer (RIPA Buffer, Wako, Osaka, Japan) containing 1% Protease Inhibitor Cocktail (NACALAI TESQUE, Kyoto, Japan). Concentrations of extracted protein samples were determined using BCA protein assay kits (Pierce, Rockford, IL, USA).

Assessment of oxidative stress and cytokine expression

Levels of 8-hydroxy-2’-deoxyguanosine (8-OHdG), an oxidative stress marker, and tumor necrosis factor-α (TNF-α) were measured using ELISA kits (8-OHdG: Japan Institute for the Control of Aging, NIKKEN SEIL Co., Shizuoka, Japan; TNF-α: R & D Systems, Minneapolis, MN, USA).

Measurements of ascorbic acid levels in cerebellum and plasma

We focused on cerebellum because 8-OHdG levels in cerebellum were noticeably increased by LISW exposure. Cerebellum tissues (60 mg) were homogenized in 5% metaphosphoric acid (840 mg) and centrifuged at 10000 g for 15 min at 4°C, essentially as previously described [42, 43]. Plasma samples were mixed with equal amount of 10% metaphosphoric acid and centrifuged at 21000 g for 10 min at 4°C. Thereafter, ascorbic acid levels of the obtained samples were measured by Hayashibara Co., using high performance liquid chromatography, prominence UFLC LC-20AD (Shimadzu Co, Kyoto, Japan).

Dihydroethidium (DHE) staining

To evaluate reactive oxygen species (ROS) formation in the tissues, cryostat sections of fresh-frozen cerebellum tissues were subjected to DHE (Invitrogen, Waltham, MA, USA) staining, essentially as previously described [11, 44]. Brains were cryosectioned (4 μm) onto glass slides, rinsed in pure distilled water for 5 minutes, and incubated in 5μM of DHE for 30 minutes in the dark. The oxidation product, ethidium, is formed from DHE by ROS resulting in ethidium accumulation within cells producing ROS. Slides were subsequently rinsed by PBS and ethidium was detected as red nuclei. Images of each section were obtained with a digital camera at a magnification of 200×. Thereafter, mean fluorescence intensity (MFI) was evaluated by measuring and averaging the immunofluorescence intensities of 10 randomly selected cells under the same conditions using the image analysis software (LuminaVision ver. 2.04, Mitani Corporation, Tokyo, Japan).

Statistics

Data are expressed as the mean ± SEM. Differences between two experimental groups were assessed using Mann–Whitney test, and those among more than three experimental groups were assessed by one-way ANOVA with the Tukey HSD post hoc test. All statistical analyses were conducted with a significance level of α = 0.05 (P < 0.05) using JMP software (version 11; SAS Institute Inc., Cary, NC).

Results

LISW-induced TBI involves oxidative stress in the brains

First, we measured levels of 8-OHdG and TNF-α to investigate the involvement of oxidative stress and inflammatory cytokine, respectively, in the brain of this LISW-induced TBI model. As shown in Fig 3A, 8-OHdG levels in cerebrum, hippocampus, and cerebellum tissues of the LISW group were all significantly higher than those of the control group. Among them, cerebellum 8-OHdG levels in the control group were undetectable, and the difference of 8-OHdG levels between the two groups was the most prominent. On the other hand, TNF-α levels did not differ between the two groups in any of the brain regions at least in the limited cases (Fig 3B). These results showed that this LISW-induced TBI model was characterized by oxidative stress in the brains and prompted us to evaluate the effects of AA2G on the model, especially focusing on cerebellar oxidative stress.

Fig 3

Levels of the following in the brain: (A) 8-hydroxy-2’-deoxyguanosine (8-OHdG) and (B) tumor necrosis factor-α (TNF-α) of the control and the LISW groups. Data are presented as the mean ± SEM (n = 4 in each group). Statistical significance was calculated by Mann–Whitney test. *P < 0.05, ns: not significant.

Post treatment with ascorbic acid does not exert significant therapeutic effects on LISW-induced TBI

The time during which the rats remained on the rod of the rotarod, which was assessed before LISW application, did not differ among the control group, the LISW group and the LISW + post AA2G group (Fig 4A). The time in the LISW group, which was assessed seven days after LISW application, was significantly shorter than that in the control group. In contrast, the time in the LISW + post AA2G group, which was assessed seven days after LISW application, tended to be longer than the LISW group; however, the difference between the two groups did not reach statistical significance (Fig 4A).

Fig 4

The time remained on the rod and 8-OHdG level in cerebellum and plasma in the LISW group and the LISW + post AA2G group.

(A) The time remained on the rod during which the rats of the laser-induced shock wave (LISW) group and LISW + post ascorbic acid 2-glucoside (post AA2G) group, evaluated before LISW application (left) and after LISW exposure (right). (B) The 8-hydroxy-2’-deoxyguanosine (8-OHdG) levels in cerebellum (left) and plasma (right) in each group. Data are presented as the mean ± SEM (n = 10 in each group). Statistical significance was calculated by one-way ANOVA with the Tukey HSD post hoc test. *p < 0.05, **p < 0.01, ns: not significant.

We also evaluated cerebellar and plasma 8-OHdG levels in the LISW and the LISW + post AA2G groups. There was no statistical significance in cerebellar 8-OHdG levels between the LISW group and the LISW + post AA2G group (Fig 4B). 8-OHdG levels in plasma also did not differ between the two groups (Fig 4B). Ascorbic acid, if administered after LISW application, showed not significant therapeutic effects on LISW-induced bmTBI.

Preventive ascorbic acid administration reduces cerebellar oxidative stress and improves motor dysfunction

As shown in Fig 5A, cerebellar ascorbic acid levels in the LISW group were significantly lower than those in the control group, whereas prophylactic administration of AA2G significantly increased the levels. Interestingly, plasma levels of ascorbic acid in the LISW group were significantly higher than those in the control group, and prophylactic administration of ascorbic acid further increased the levels (Fig 5A).

Fig 5

AA levels in cerebellum and plasma, 8-OHdG levels in cerebellum and plasma, DHE staining of cerebellum, and the time remaining on the rod in the control group, the LISW group, and the LISW + pre and post AA2G group.

(A) Ascorbic acid (AA) levels and in cerebellum (upper) and plasma (lower) of the control, laser-induced shock wave (LISW), and LISW + pre and post ascorbic acid 2-glucoside (AA2G) groups. (B) 8-hydroxy-2’-deoxyguanosine (8-OHdG) in cerebellum (upper) and plasma (lower) of each group. (C) Representative photomicrographs of dihydroethidium (DHE) staining of cerebellum from each group (upper). Relative fluorescence intensity of each group (lower). (D) The time during which the rats of each group remained on the rod, evaluated before LISW application (left) and after LISW exposure (right). Data are presented as the mean ± SEM (A and C; n = 4 in each group, B and D; n = 10 in each group). Statistical significance was calculated by one-way ANOVA with the Tukey HSD post hoc test. *p < 0.05, **p < 0.01, ns: not significant.

Both cerebellar and plasma 8-OHdG levels in the LISW group were significantly higher than those in the control group; however, these levels were significantly decreased in the LISW + pre and post AA2G group (Fig 5B). These findings were supported by the results of DHE staining; ROS formation in cerebellum of the LISW group was significantly increased as compared to that of the control group, whereas the levels of ROS formation were significantly reduced in the LISW + pre and post AA2G group (Fig 5C).

Fig 5D shows the time during which the rats of each group remained on the rod of the rotarod. Although there was no statistical difference in the time among the three groups before LISW application (Fig 5D), the time in the LISW group, which was assessed seven days after LISW application, was significantly shorter than that in the control group (Fig 5D). On the other hand, the time in the LISW + pre and post AA2G group was significantly longer as compared to that in the LISW group. These results showed that prophylactic administration of AA2G increased cerebellar ascorbic acid levels, reduced ROS formation and resultant oxidative stress in cerebellum, and mitigated motor dysfunction of LISW-induced bmTBI in rats.

Discussion

The present study demonstrated that prophylactic AA2G administration ameliorated the motor coordination disorder due to the LISW-induced TBI (Fig 5D) through attenuation of the oxidative stress in the cortex and cerebellum (Fig 3A). Studlack et al. previously used beam walk, accelerating rotarod, rearing, open field, elevated plus maze, and light–dark box task tests in bTBI injury model rats to evaluate their motor function and anxiety-related behavior. They observed that the accelerating rotarod test exhibited significant differences between the bTBI and control groups from day 1 to day 14 post injury [45]. We considered that the prolonged symptoms were suitable for evaluating the efficacy of AA2G and confirmed that the accelerating rotarod test exhibited significant differences between the LISW and control groups in the present study. Expectedly AA2G restored the loss of cerebellar ascorbic acid levels, which was observed in the LISW group (Fig 5A). We examined the levels of TNF-α, one of the representative inflammatory cytokines in neuronal inflammation derived from monocytes and macrophages [8], in the brain. Although involvement of inflammatory pathway in the pathogenesis of bmTBI has been suggested [8], TNF-α levels in the brain were not elevated by LISW application in the present study (Fig 3B). The timing of TNF-α measurement might have affected the results.

Ascorbic acid levels in cerebellum of the LISW group were significantly lower as compared to those of the control group, whereas the levels in plasma of the LISW group were significantly higher than those of the control group (Fig 5A). There seemed to be a discrepancy between local and systemic ascorbic acid levels. In this regard, we considered a possibility that ascorbic acid biosynthesis was increased in the LISW group to compensate the loss of brain ascorbic acid. However, it should be noted that such compensation cannot be applicable to humans, because rats can synthesize ascorbic acid in vivo but the capacity is lost in humans due to mutations in the gene encoding the last enzyme in ascorbic acid biosynthesis pathway [46].

Previous reports have demonstrated that the mechanisms of bTBI are unique and complex [24, 47–50]. Subsequent to brain damage that occurs at the immediate moment of exposure, the secondary effects yield a spectrum of injuries. It includes ROS production causing cellular damage through oxidation of cellular molecules such as DNA, proteins and lipids, which contributes to alterations in protein conformation and binding [11]. 8-OHdG is a stable product of oxidatively damaged DNA formed by hydroxyl radical, singlet oxygen and direct photodynamic action, and can be detected in tissue, serum, urine and other biomaterials [51]. Indeed, in the present study, the levels of 8-OHdG were significantly elevated by LISW application. This ROS production may occur not only as a direct consequence of bmTBI but also as a result of secondary injury. For example, it was reported that the hemoglobin released from the disrupted red blood cells after blast exposure catalyzed free radical formation [52]. Further, the levels of oxidative stress and endogenous antioxidant proteins, such as superoxide dismutase, increased within several hours after blast exposure, and returned to normal levels within 5 days [24]. In addition, the elevation in the levels was directly proportional to the severity of injury [24]. Our previous study showed that single application of an LISW to the brain caused spreading depolarization, vasoconstriction and prolonged severe hypoxia in the cortex, which can induce neuronal cell alterations [21].

Ascorbic acid is a potent water soluble antioxidant in biological fluid [26] and has strong advantages for clinical application due to easy availability, low-cost, and minimal adverse effects. Indeed, various reports have shown its usefulness as a powerful antioxidant. For example, preventive and therapeutic potential of ascorbic acid in neurodegenerative diseases or chronic inflammatory diseases was reported [53, 54]. Administration of ascorbic acid also rescued irradiated mice from radiation-induced lethal gastrointestinal damage and reduced their mortality [41, 42, 55]. Importantly, no serious adverse effects related to ascorbic acid administration were reported in these studies. Further, AA2G is an ascorbic acid derivative that is stable in aqueous solution and barely induces cellular toxicity in cultured stem-cells, unlike ascorbic acid [56]. In addition, AA2G has protective effects against Helicobacter pylori infection in gastric epithelial cells [30] and confers protective effects on human sperm motility preservation through the freeze–thaw cycle [57]. In the present study, we evaluated the efficacy of AA2G in experimental LISW-induced TBI in rats at a dose of 250 mg/kg/day (this amount of AA2G contains 125 mg/kg/day of ascorbic acid). The dose of ascorbic acid was less than those had been used in the previous studies, demonstrating that ascorbic acid administration rescued irradiated mice [41, 42, 55]. However, because AA2G is hard to be resolved and considered to be more stable than conventional ascorbic acid [30], we chose our dosing at this level. Ascorbic acid effectively scavenges free radicals in vitro and might therefore be used as a radio-protectant that effectively scavenges ROS formed during radiation exposure [58]. There have been many reports regarding the protective effects of antioxidants given prior to exposure; however, there are few studies of successful post-exposure treatment [43, 59]. In present study, AA2G did not exert significant therapeutic effects on LISW-induced TBI when it was administered after LISW application; motor function assessed by rotarod apparatus and levels of oxidative stress did not significantly differ between the LISW and the LISW + post AA2G groups (Fig 4A). In line with this finding, in our previous studies [42, 43], treatment with ascorbic acid after radiation exposure could not also rescue lethal gastrointestinal damage in mice. In these studies, pre-treatment or post-treatment alone did not improve the survival of the mouse model; however, pre- and post-treatment significantly improved the survival. It is possible that scavenging ROS generated immediately after radiation by boosting the pretreatment with ascorbic acid may be necessary to improve the survival of irradiated mice. However, additional post-treatment with ascorbic acid also may be indispensable to further improve survival due to late or ongoing damage by oxidative stress.

We focused on cerebellar oxidative stress because 8-OHdG levels in cerebellum were noticeably increased by LISW exposure. Although the reason of the largest increase in cerebellar oxidative stress remains to be solved, motor coordination disorder, which can be associated with damage to the motor area of cortex and cerebellar, was actually observed in the LISW group. However, motor dysfunction is just a part of various symptoms which are induced by LISW-induced TBI. The involvement of oxidative stress and the efficacy of ascorbic acid in other symptoms of LISW-induced TBI should be carefully investigated.

In the present study, LISWs were applied only to the brain and were not accompanied by dynamic pressure. Therefore, it can be concluded that these results show the effects of the primary mechanism solely on the brain. Use of a shock tube would not have enabled us to completely exclude the effects of dynamic pressure or exposure to other part of the body; therefore, our results are useful to understand the mechanisms of primary bTBI. However, careful attention should be paid to the differences in characteristics between LISWs and actual blasts. Therefore, a comparative experimental study using a shock tube could give valuable insight into the mechanism and possible intervention for bTBI.

Limitations

In the present study, there was only experimental data obtained at 7 days after LISW exposure, and longer follow-up was not done. The present study only used male and 10-week-old rats.

This study was performed based on the assumption that the impulse was the primary parameter to determine LISW-induced TBI. We recently identified methods to control the duration of LISW and are currently conducting experiments to examine the validity of this assumption. Therefore, the results obtained in the present study will be reevaluated after the validation.

Conclusions

The present study showed that LISW-induced TBI involved ROS formation in the cerebellum and that prophylactic AA2G administration attenuated oxidative stress. In addition, prophylactic AA2G administration significantly ameliorated the motor coordination disorder induced by LISW-induced TBI. In contrast, therapeutic AA2G administration showed no significant effects. Our results indicated the oral AA2G could be a promising prophylactic agent for shock wave-related TBI.

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21 Nov 2019

PONE-D-19-25644

Oral ascorbic acid 2-glucoside prevents coordination disorder induced by blast-related mild traumatic brain injury in a rat model

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: 'Oral ascorbic acid 2-glucoside prevents coordination disorder induced by blast-related mild traumatic brain injury in a rat model'. This is a well structred and well written manuscript. It is acceptable in its current form.

Reviewer #2: Comments

Introduction:

“Lines 62-65: Notably, most of the bTBI patients lack any external physical evidences and

abnormalities detected by conventional imaging devices, but in the chronic phase, they

develop persistent physiological and psychological changes associated with

higher-order brain dysfunction, which is called blast-related mild TBI (bmTBI) (4, 5).”

In reference to this statement, authors should specify that most of the bTBI patients who are exposed to low or mild blast exposure may not show signs of overt pathology in the acute phase. Mild blast should not be confused with mild TBI.

Lines 66-70: The cited work primarily evaluated the effects of shock wave (blast overpressure and/or underpressure) on neuropathology, oxidative stress, and the applicability of antioxidants in ameliorating the behavioral deficits. The authors are applying these findings to laser-induced (LISW) brain injury. The authors need to establish the relevance of LISW to blast overpressure.

In general, the introduction is too short. I suggest the following:

1) Include literature that compared the common data elements of LISW and blast shock wave.

2) Include literature that shows LISW can have the same effects as IEDs, microexplosions and blast/shock tubes to be able to make a strong case for calling this model of injury a blast model.

3) Cite work from other authors who have shown blast related oxidative stress and inflammation. A single sentence on BBB disruption, inflammation, and edema doesn’t do justice to the body of literature that exists on blast-related neurological deficits. Since the focus is on oxidative stress, the authors can include few sentences on ROS, RNS, free radicals etc. There should be referece to some prior work that shows behavioral/motor deficits after blast. Also ref # 7 focuses on the efficacy of increasing the cerebral volume in being protective against blast-induced TBI.

4) How have antioxidants helped against blast-TBI? Authors have mentioned free radical scavenging. What happens to the endogenous antioxidatve mechanisms after blast exposure(s)?

5) Why AA2G in particular? What preliminary studies or literature supports the use of this particular antioxidant?

6) Why rotarod test in particular?

“AA2G (Hayashibara Co., Okayama, Japan) was dissolved in distilled water and was administered daily per os (p.o.) to rats at 250 mg/kg/15mL/day (ascorbic acid: 125 mg/kg/day) after LISW application for a total of 7 days (LISW + post AA2G group).” Did the animals receive ascorbic acid and its derivative (AA2G)? Please clarify the dosage and the daily frequency (once/day?) of what was administered.

Animal grouping: The description is a bit confusing. It has been described differently in different parts of the manuscript. Figure 1 is helpful I understanding the grouping. It seems like a total of four animal groups were used, which are: 1) Control, 2) LISW, 3) LISW+pre AA2G, and 4) LISW+pre-and post- AA2G. Figure 1B lists LISW again. Did the experimental design have a total of 20 animals in LISW group (combining Figures 1 A and B)? If so, why?

Why was rotarod testing done at 1 day prior to injury in some groups and 3 days prior to injury in other groups?

Generation and Characteristics of LISW: The shock wave generated by lasers (Fig 2) lack the underpressure that’s seen in the primary blast wave. It seems the authors are establishing LISW as a model of simulated blast injury. The authors need to provide more details (common data elements) of the pressure profile itself. Figure 2B- What are the characteristics after the 2 microsecond period? Are there any reflections or is it a flat line? Is the aim here to simulate primary blast wave? One has to make a really strong case to compare LISWs to blast explosions- otherwise the title is misleading. The application of the injury is very focal which is in contrast to blast injury (that’s diffused). Simulated blast pressures include static and dynamic pressures, which is lacking in the LISW model of injury. I strongly suggest changing the title of the manuscript to indicate this distinction clear.

“Although peak pressures and durations of LISWs are roughly three orders of magnitude higher and three orders of magnitude shorter than those of medically relevant actual explosions, respectively (28), impulse (time-integrated positive pressures) of actual explosions and LISWs used in this study are in the same order.” Please clarify. What blast pressures do they intend to compare the generated LISWs. If the comparison is made with mild blast intensities (10-15 PSI), then an increase by three orders of magnitude in peak intensity would make sense. What are the impulse values of the positive phase of LISW pressure profile?

“… impulse (time-integrated positive pressures) of actual explosions and LISWs used in this study are in the same order: The comment on impulse may not be correct. Please provide relevant impulse values from the literature.

“The laser spot size and fluence on the target were kept constant at 3 mm and 2.4 J/cm2, respectively, producing an LISW with an impulse of 54 Pa•s.” Please provide more details of the peak, duration, frequency, power, etc?

In general, LISW model as presented here may not be comparable to blast explosions. One would err on the side of caution and call this model simply as LISW model instead of clubbing it under blast. There isn’t enough evidence presented in the manuscript to support the author’s claim.

Line 155: rpm/min- correct to rpm

Statistics: Was the data checked for normal distribution?

Results:

Figure 3: Why are the sample sizes small? N= 4? With such small sample size, what’s of the power of the design? Is there any explanation for TNF-α being insignificantly different between control and injury groups?

Figures 4A and 4B should include the control group as well.

It’s been shown that post-LISW administration of AA2G is not beneficial in circumventing the oxidative stress induced by the injury. Pre+post- AA2G administration seems to be beneficial. Is it possible that simply the preemptive treatment with AA2G might be enough to inhibit the progression of oxidative stress? Why haven’t authors chosen to study only the prophylactic properties of the antioxidant? Please provide a justification. Combining the pre-and post- application in the same group of animals doesn’t necessarily rule out the therapeutic properties of AA2G- it suggests a combined prophylactic and therapeutic potential of AA2G.

**********

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Reviewer #1: Yes: Pinar Kuru Bektasoglu

Reviewer #2: Yes: Usmah Kawoos

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30 Dec 2019

Response to Reviewer #2:

We would like to express our sincerest gratitude to the reviewer who identified areas of the manuscript that required revisions or modifications, and also for their insightful comments that have helped us to significantly improve our manuscript. We believe that laser-induced shock waves (LISWs) could be a useful tool to investigate the primary mechanisms of blast-related traumatic brain injury (bTBI) because they are spatially defined and are not accompanied by dynamic pressure, enabling site-selective analysis of primary injury mechanism. However, as highlighted by the reviewer, there are considerable differences in the characteristics of LISWs and actual blasts. Thus, the present animal model was changed and renamed as “LISW-induced TBI model,” rather than a “blast TBI model”. We have clearly stated our rationale for using this model (impulse mimicking) in the revised manuscript and have also discussed the LISW conditions compared with blasts as previously described by Mishra et al. (Sci. Rep., 2016). Our specific responses to each comment are detailed below.

Comment 1. “Lines 62-65: Notably, most of the bTBI patients lack any external physical evidences and abnormalities detected by conventional imaging devices, but in the chronic phase, they develop persistent physiological and psychological changes associated with higher-order brain dysfunction, which is called blast-related mild TBI (bmTBI) (4, 5).”

In reference to this statement, authors should specify that most of the bTBI patients who are exposed to low or mild blast exposure may not show signs of overt pathology in the acute phase. Mild blast should not be confused with mild TBI.

Response: Thank you for your comment. We have revised the following text (page 5, lines 62–66) from “Notably, most of the bTBI patients lack any external physical evidences and abnormalities detected by conventional imaging devices, but in the chronic phase, they develop persistent physiological and psychological changes associated with higher-order brain dysfunction, which is called blast-related mild TBI (bmTBI) [4, 5]” to “Notably, most bTBI patients who are exposed to low or mild blast exposure lack any external physical evidences and abnormalities detected by conventional imaging devices; therefore, they are being diagnosed as having blast-related mild TBI (bmTBI). However, during the chronic phase, they develop persistent physiological and psychological changes associated with higher-order brain dysfunction during the chronic phase [4, 5].”

Comment 2. Lines 66-70: The cited work primarily evaluated the effects of shock wave (blast overpressure and/or underpressure) on neuropathology, oxidative stress, and the applicability of antioxidants in ameliorating the behavioral deficits. The authors are applying these findings to laser-induced (LISW) brain injury. The authors need to establish the relevance of LISW to blast overpressure.

Response: As described above, we have revised the manuscript considering the difference in characteristics between LISWs and actual blasts. In the present study, we found that topical application of LISWs to the rat brain caused oxidative stress and motor dysfunctions. There are some similarities in symptoms with those reported for mild bTBI rodent models using a shock tube [37, 38].

2-1) Include literature that compared the common data elements of LISW and blast shock wave.

Response: We have included new references [37] and [38] to discuss the LISW conditions used in this study compared with blast shock waves.

2-2) Include literature that shows LISW can have the same effects as IEDs, microexplosions and blast/shock tubes to be able to make a strong case for calling this model of injury a blast model.

Response: We have renamed the present model to an “LISW-induced TBI model,” rather than a “blast TBI model” to account for the characteristic differences between LISWs and actual blasts. In the present study, we assumed that the impulse was the primary parameter for determining brain injury. A description of the assumption has been included to the Materials and Methods section. While the impulse of LISWs used in the present study was low (54 Pa•s), we were unable to demonstrate that the TBI in our model was mild. Thus, the word “mild” has been deleted.

2-3) Cite work from other authors who have shown blast related oxidative stress and inflammation. A single sentence on BBB disruption, inflammation, and edema doesn’t do justice to the body of literature that exists on blast-related neurological deficits. Since the focus is on oxidative stress, the authors can include few sentences on ROS, RNS, free radicals etc. There should be referece to some prior work that shows behavioral/motor deficits after blast. Also ref # 7 focuses on the efficacy of increasing the cerebral volume in being protective against blast-induced TBI.

Response: We apologize for the lack of a detailed explanation about blast-related oxidative stress. As per the reviewer’s comment, we have included the following text (page 5, line 16 to page 6, line 10): “Oxidative stress has been implicated in multiple models of TBI [12, 13] and is mainly induced by reactive oxidative species (ROS) such as superoxide, hydroxyl radical, and hydrogen peroxide [14, 15]. While basal levels of ROS are present during normal redox reactions and the electron transport chain, excess amounts, as seen after injury, can be harmful. NADPH oxidase (NOX) is a multi-subunit enzyme that catalyzes the formation of superoxide radicals from available molecular oxygen. NOX is upregulated in multiple brain regions following blast injury, and neurons maximally contributes to a higher increase in the hippocampus compared with other neural cells [16, 17]. Moreover, oxidative stress also contributes to enhanced BBB permeability during bTBI via a pathway that involves increased matrix metalloproteinase activation [18].”

2-4) How have antioxidants helped against blast-TBI? Authors have mentioned free radical scavenging. What happens to the endogenous antioxidatve mechanisms after blast exposure(s)?

Response: As the reviewer has pointed out, the mechanisms of endogenous antioxidation are very important. Therefore, we have added the following text (page 6, lines 11–16 and page 7, lines 1-3): “Hydrogen sulfide, which is one of the antioxidants, also affected the controlled cortical impact injury in rats, leading to improved neurologic dysfunction, increased activities of endogenous antioxidant enzymes (superoxide dismutase and catalase), decreased levels of oxidative products (malondialdehyde and 8-iso-prostaglandin F2α), increased BBB permeability, and attenuation of brain edema. Furthermore, the use of the KATP channel blocker, 5-hydroxydecanoate, further demonstrated activation of mitochondrial adenosine triphosphate sensitive potassium channels, and oxidative stress was reduced following treatment with exogenous hydrogen sulfide [19, 20].”

2-5) Why AA2G in particular? What preliminary studies or literature supports the use of this particular antioxidant?

Response: Thank you for highlighting this. We apologize for not acknowledging the efficacy of AA2G. We have included the following text (page 26, lines 6–10): “Further, AA2G is an ascorbic acid derivative that is stable in aqueous solution and barely induces cellular toxicity in cultured stem cells, unlike ascorbic acid [56]. In addition, AA2G has protective effects against Helicobacter pylori infection in gastric epithelial cells [30] and confers protective effects on human sperm motility preservation through the freeze–thaw cycle [57] .”

2-6) Why rotarod test in particular?

Response: We apologize for not explaining our rationale for selecting the rotarod test. As per the reviewer’s comment, we have added the following text (page 23 lines 5–12): “Studlack et al. previously used beam walk, accelerating rotarod, rearing, open field, elevated plus maze, and light–dark box task tests in bTBI injury model rats to evaluate their motor function and anxiety-related behavior. They observed that the accelerating rotarod test exhibited significant differences between the bTBI and control groups from day 1 to day 14 post injury [45]. We considered that the prolonged symptoms were suitable for evaluating the efficacy of AA2G and confirmed that the accelerating rotarod test exhibited significant differences between the LISW and control groups in the present study.”

Comment 3. “AA2G (Hayashibara Co., Okayama, Japan) was dissolved in distilled water and was administered daily per os (p.o.) to rats at 250 mg/kg/15mL/day (ascorbic acid: 125 mg/kg/day) after LISW application for a total of 7 days (LISW + post AA2G group).” Did the animals receive ascorbic acid and its derivative (AA2G)? Please clarify the dosage and the daily frequency (once/day?) of what was administered.

Response: We apologize for this confusion. In the present study, 250 mg of AA2G contained 125 mg of ascorbic acid. AA2G was administered daily in the drinking water. We modulated the concentration of AA2G in the drinking water by measuring the daily water intake to administer a dose of 250 mg/kg/day of AA2G to the rats.

We have revised the following text in lines 12–15 on page 8 from “AA2G (Hayashibara Co., Okayama, Japan) was dissolved in distilled water and was administered daily per os (p.o.) to rats at 250 mg/kg/15mL/day (ascorbic acid: 125 mg/kg/day) after LISW application for a total of 7 days (LISW + post AA2G group)” to “AA2G (Hayashibara Co., Okayama, Japan) was dissolved in distilled drinking water and was administered daily per os (p.o.) to rats at a dose of 250 mg/kg/day (250 mg of AA2G contained 125 mg/kg/day of ascorbic acid) after LISW application for a total of 7 days (LISW + post AA2G group).”

Comment 4. Animal grouping: The description is a bit confusing. It has been described differently in different parts of the manuscript. Figure 1 is helpful I understanding the grouping. It seems like a total of four animal groups were used, which are: 1) Control, 2) LISW, 3) LISW+pre AA2G, and 4) LISW+pre-and post- AA2G. Figure 1B lists LISW again. Did the experimental design have a total of 20 animals in LISW group (combining Figures 1 A and B)? If so, why?

Response: Thank you for highlighting this. The experimental design included a total of 20 animals in the LISW group. We observed no significant difference between the LISW and LISW + post-AA2G groups, and we could only add pre- and post-AA2G groups as further experiment. We would like to compare LISW group and pre- and post-AA2G group under the same conditions because ascorbic acid levels in the tissue were quite unstable. Therefore, we could perform additional experiments (assessment of motor function, oxidative stress, ascorbic acid levels, and DHE staining) to compare the control, LISW, and LISW + pre- and post-groups.

Comment 5. Why was rotarod testing done at 1 day prior to injury in some groups and 3 days prior to injury in other groups?

Response: We apologize for the confusion in the description of the method. We have revised Fig. 1 and the following text in line 11 on page 9 from “their motor function was assessed using a rotarod apparatus 1 day before and 7 days after the procedures” to “their motor function was assessed using a rotarod apparatus 3 day before and 7 days after the procedures.”

Figure 1 (revised)

Comment 6. Generation and Characteristics of LISW: The shock wave generated by lasers (Fig 2) lack the underpressure that’s seen in the primary blast wave. It seems the authors are establishing LISW as a model of simulated blast injury. The authors need to provide more details (common data elements) of the pressure profile itself. Figure 2B- What are the characteristics after the 2 microsecond period? Are there any reflections or is it a flat line? Is the aim here to simulate primary blast wave? One has to make a really strong case to compare LISWs to blast explosions- otherwise the title is misleading. The application of the injury is very focal which is in contrast to blast injury (that’s diffused). Simulated blast pressures include static and dynamic pressures, which is lacking in the LISW model of injury. I strongly suggest changing the title of the manuscript to indicate this distinction clear.

Response: Typical LISWs are dominated by positive pressure, although negative pressure can be generated under certain conditions. In the present study, generated LISWs did not interact with the external environment and were directly propagated into the rat brain. There were no pressure signals beyond the time range shown in Fig. 2B (>2 μs). LISWs are spatially confined and lack dynamic pressure component resulting in accelerating injury (the tertiary mechanism). We believe that these characteristics of LISWs provide a unique tool that can be used to analyze the primary mechanisms of bTBI. However, we agree that LISWs and accrual blasts differ in characteristics. Thus, we renamed our model as an “LISW-induced TBI model”, rather than a “blast TBI model”. The LISW impulse used was as low as 54 Pa•s, but the peak overpressure was high. Therefore, we have changed the title to remove the word “mild”.

Comment 7. “Although peak pressures and durations of LISWs are roughly three orders of magnitude higher and three orders of magnitude shorter than those of medically relevant actual explosions, respectively (28), impulse (time-integrated positive pressures) of actual explosions and LISWs used in this study are in the same order.” Please clarify. What blast pressures do they intend to compare the generated LISWs. If the comparison is made with mild blast intensities (10-15 PSI), then an increase by three orders of magnitude in peak intensity would make sense. What are the impulse values of the positive phase of LISW pressure profile?

Comment 8. “… impulse (time-integrated positive pressures) of actual explosions and LISWs used in this study are in the same order: The comment on impulse may not be correct. Please provide relevant impulse values from the literature.

Response:

Responses to Comments 7 & 8:

We have included two references (new ref. [37], [38]) to describe the pressure characteristics of improvised explosive devices (IEDs), and we have described the LISW conditions with reference to that reported by Mishra et al. [17] in the Materials and Methods section. The phrases “three orders of magnitude” has been corrected and changed to “two to three orders of magnitude.” We assumed that the impulse was the primary parameter to determine brain injury and used LISWs replicating the impulse of a typical IED.

In accordance with the reviewer’s comment, we have included the following text (page 10, line 3 to page 11, line 5): “No dynamic pressure is produced in the generation of LISWs; therefore, the effects of acceleration or displacement can be excluded in animal studies, thus enabling analysis of the primary mechanism of bTBI. Positive pressure duration (hereafter simply termed duration) is an important parameter to examine shock wave to brain interactions. The duration of a typical IED explosion-related shock wave ranges from 200 μs to several milliseconds [37, 38]; however, the interaction of the human brain with such IED-related shock waves, especially brain to skull boundary effects, cannot be reproduced in the brains of small animals when using similar duration shock waves. Therefore, a scaling law that considers anatomical differences between human and animal brains should be used. However, few studies have focused on this important issue. In the present study, we assumed that the impulse (time-integrated positive pressure component) of the shock wave was the primary parameter for determining brain injury. Alley previously estimated impulses of various types of IEDs as a function of propagation distance ranging from ~6.9 to ~100 Pa•s for propagation distances of 1–10 m [37]. Although the duration of LISWs is approximately two to three orders of magnitude shorter than that of typical IED shock waves, their impulses can be easily controlled by changing the laser fluence and hence the peak pressure of LISW. IED impulses within this range can be replicated using LISWs. The limitations associated with this assumption are described in Discussion and Limitations section.”

We have also added the following text (page 12, lines 6-12): “Mishra et al. examined physiological and pathological changes, as well as changes in blood-borne biomarkers in rats exposed to blasts with a wide range of overpressure and impulses using a shock tube [17]. They determined the conditions under which mild, moderate, severe, and lethal bTBIs were induced, and postulated that mild bTBI was caused by blasts with ≤145 kPa, with a corresponding impulse of 250 Pa•s. The impulse used in this study (54 Pa•s) was within this range.”

Comment 9. “The laser spot size and fluence on the target were kept constant at 3 mm and 2.4 J/cm2, respectively, producing an LISW with an impulse of 54 Pa•s.” Please provide more details of the peak, duration, frequency, power, etc?

Response: The temporal pressure profile of the used LISW is shown in Fig. 2B (blue line). A single pulse of LISW was applied to each site on the skull.

Comment 10. In general, LISW model as presented here may not be comparable to blast explosions. One would err on the side of caution and call this model simply as LISW model instead of clubbing it under blast. There isn’t enough evidence presented in the manuscript to support the author’s claim.

Response: We agree with the reviewer’s comment and have changed the name of our model to an “LISW-induced TBI model”.

Comment 11. Line 155: rpm/min- correct to rpm

Response: We apologize for this error. We have changed “rpm/min” to “rpm” in line 5 on page 14.

Comment 12. Statistics: Was the data checked for normal distribution?

Response: We apologize for not verifying the normal distribution of our data. We performed Kolmogorov–Smirnov test to evaluate whether our data was normally distributed and confirmed that the data in Figs. 4 and 5 were normally distributed. In contrast, we were unable to perform a Kolmogorov–Smirnov test on the data in Fig. 3 because of the small sample size. Therefore, we performed Mann–Whitney test instead of a Student’s t test to analyze the data in Fig. 3. We have revised the following text in line 9 on page 17 from “Differences between two experimental groups were assessed using Student’s t-test” to “Differences between two experimental groups were assessed using Mann–Whitney test.”

Comment 13. Figure 3: Why are the sample sizes small? N= 4? With such small sample size, what’s of the power of the design? Is there any explanation for TNF-α being insignificantly different between control and injury groups?

Response: As highlighted by the reviewer, the small sample sizes in Fig. 3 meant that we were unable to draw a significant conclusion. However, this experiment was a preliminary step in our study and was not designed to be conclusive. We did not focus on inflammatory cytokines in this figure, but instead, we examined oxidative stress. This was performed to demonstrate that oxidative stress in the brain may increase and TNF-α may not significantly increase. Furthermore, there was a significant difference in the oxidative stress levels between the control and LISW groups in this setting, and there was no need to add additional experiments. We have added the following text (page 18, line 10): “at least in the limited cases.”

Comment 14. Figures 4A and 4B should include the control group as well.

Response: Thank you for highlighting this error. We have included the control group to Figs. 4A and 4B. Statistical analyses were conducted using one-way ANOVA with Tukey HSD post hoc test. We found that there was no significant difference between the LISW and post AA2G groups.

Fig 4 (revised)

Comment 15. It’s been shown that post-LISW administration of AA2G is not beneficial in circumventing the oxidative stress induced by the injury. Pre+post- AA2G administration seems to be beneficial. Is it possible that simply the preemptive treatment with AA2G might be enough to inhibit the progression of oxidative stress? Why haven’t authors chosen to study only the prophylactic properties of the antioxidant? Please provide a justification. Combining the pre-and post- application in the same group of animals doesn’t necessarily rule out the therapeutic properties of AA2G- it suggests a combined prophylactic and therapeutic potential of AA2G.

Response: We apologize for not including a detailed description of the AA2G administration. In our previous study on the effects of ascorbic acid administration in a mouse model of radiation-induced gastrointestinal damage (Ito et al, Int J Mol Sci 2013, 14 19168-19635), pre-treatment alone or post-treatment alone did not improve the survival of the mice. Nevertheless, in the present study, pre and post-treatment significantly improved the survival of the mice. It is possible that scavenging of ROS generated immediately after radiation by pretreatment with ascorbic acid may be necessary to improve the survival of irradiated mice. However, additional post-treatment with ascorbic acid also may be indispensable for further improving the survival due to late or ongoing damage by oxidative stress. Oxidative stress may also be an important factor in bmTBI injury; therefore, pre + post-treatment by AA2G may have been more effective than pre-treatment alone. For this reason, we chose pre + post-treatment model instead of pre-treatment model.

We have included the following text (page 27, lines 10-16): “In these studies, pre-treatment or post-treatment alone did not improve the survival of the mouse model; however, pre- and post-treatment significantly improved the survival. It is possible that scavenging of ROS generated immediately after radiation by boosting the pretreatment with ascorbic acid may be necessary to improve the survival of irradiated mice. However, additional post-treatment with ascorbic acid may also be indispensable to further improve the survival due to late or ongoing damage by oxidative stress.”

We hope that our responses and revisions have appropriately addressed your comments, and that our revised manuscript is now suitable for publication. We look forward to hearing from you. Please let us know if you have any further questions or requirements to improve our manuscript.

Submitted filename: Response_to_Reviewer.docx

Click here for additional data file.

10 Mar 2020

Oral ascorbic acid 2-glucoside prevents coordination disorder induced via laser-induced shock waves in rat brain

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16 Mar 2020

PONE-D-19-25644R1

Oral ascorbic acid 2-glucoside prevents coordination disorder induced via laser-induced shock waves in rat brain

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PLOS ONE Editorial Office Staff

on behalf of

Dr. Alfred S Lewin

Section Editor

PLOS ONE