Treatment of traumatic brain injury with 17α-ethinylestradiol-3-sulfate in a rat model

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OBJECTIVE

17α-ethynylestradiol-3-sulfate (EE-3-SO4) is a highly water-soluble synthetic estrogen that has an extended half-life (∼ 10 hours) over that of naturally occurring estrogen (∼ 10 minutes). In this study, EE-3-SO4 was evaluated in a lateral fluid percussion–induced traumatic brain injury (TBI) model in rats.

METHODS

A total of 9 groups of Sprague-Dawley rats underwent craniectomy. Twenty-four hours later, lateral fluid percussion was applied to 6 groups of animals to induce TBI; the remaining 3 groups served as sham control groups. EE-3-SO4 (1 mg/kg body weight in 0.4 ml/kg body weight) or saline (vehicle control) was injected intravenously 1 hour after TBI; saline was injected in all sham animals. One day after EE-3-SO4/saline injection, intracranial pressure (ICP), cerebral perfusion pressure (CPP), and partial brain oxygen pressure (PbtO2) were measured in Groups 1–3 (2 TBI groups and 1 sham group), and brain edema, diffusion axonal injury, and cerebral glycolysis were assessed in Groups 4–6 using MRI T2 mapping, diffusion tensor imaging (DTI), and FDG-PET imaging, respectively. Four days after dosing, the open-field anxiety of animals was assessed in Groups 7–9 by measuring the duration that each animal spent in the center area of an open chamber during 4 minutes of monitoring.

RESULTS

EE-3-SO4 significantly lowered ICP while raising CPP and PbtO2, compared with vehicle treatment in TBI-induced animals (p < 0.05). The mean size of cerebral edema of TBI animals treated with EE-3-SO4 was 25 ± 3 mm3 (mean ± SE), which was significantly smaller than that of vehicle-treated animals (67 ± 6 mm3, p < 0.001). Also, EE-3-SO4 treatment significantly increased the fractional anisotropy of the white matter in the ipsilateral side (p = 0.003) and cerebral glycolysis (p = 0.014). The mean duration that EE-3-SO4-treated animals spent in the center area was 12 ± 2 seconds, which was significantly longer than that of vehicle-treated animals (4 ± 1 seconds; p = 0.008) but not different from that of sham animals (11 ± 3 seconds; p > 0.05).

CONCLUSIONS

These data support the clinical use of EE-3-SO4 for early TBI treatment.

ABBREVIATIONS CPP = cerebral perfusion pressure; DAI = diffuse axonal injury; DTI = diffusion tensor imaging; EE= 17α-ethinyl estradiol; EE-3-SO4 = 17α-ethynylestradiol-3-sulfate; E2 = 17β-estradiol; E2-SO4 = 17β-estradiol sulfate; ICP = intracranial pressure; PbtO2 = partial brain oxygen pressure; SUV = standardized uptake value; TBI = traumatic brain injury.

Abstract

OBJECTIVE

17α-ethynylestradiol-3-sulfate (EE-3-SO4) is a highly water-soluble synthetic estrogen that has an extended half-life (∼ 10 hours) over that of naturally occurring estrogen (∼ 10 minutes). In this study, EE-3-SO4 was evaluated in a lateral fluid percussion–induced traumatic brain injury (TBI) model in rats.

METHODS

A total of 9 groups of Sprague-Dawley rats underwent craniectomy. Twenty-four hours later, lateral fluid percussion was applied to 6 groups of animals to induce TBI; the remaining 3 groups served as sham control groups. EE-3-SO4 (1 mg/kg body weight in 0.4 ml/kg body weight) or saline (vehicle control) was injected intravenously 1 hour after TBI; saline was injected in all sham animals. One day after EE-3-SO4/saline injection, intracranial pressure (ICP), cerebral perfusion pressure (CPP), and partial brain oxygen pressure (PbtO2) were measured in Groups 1–3 (2 TBI groups and 1 sham group), and brain edema, diffusion axonal injury, and cerebral glycolysis were assessed in Groups 4–6 using MRI T2 mapping, diffusion tensor imaging (DTI), and FDG-PET imaging, respectively. Four days after dosing, the open-field anxiety of animals was assessed in Groups 7–9 by measuring the duration that each animal spent in the center area of an open chamber during 4 minutes of monitoring.

RESULTS

EE-3-SO4 significantly lowered ICP while raising CPP and PbtO2, compared with vehicle treatment in TBI-induced animals (p < 0.05). The mean size of cerebral edema of TBI animals treated with EE-3-SO4 was 25 ± 3 mm3 (mean ± SE), which was significantly smaller than that of vehicle-treated animals (67 ± 6 mm3, p < 0.001). Also, EE-3-SO4 treatment significantly increased the fractional anisotropy of the white matter in the ipsilateral side (p = 0.003) and cerebral glycolysis (p = 0.014). The mean duration that EE-3-SO4-treated animals spent in the center area was 12 ± 2 seconds, which was significantly longer than that of vehicle-treated animals (4 ± 1 seconds; p = 0.008) but not different from that of sham animals (11 ± 3 seconds; p > 0.05).

CONCLUSIONS

These data support the clinical use of EE-3-SO4 for early TBI treatment.

Traumatic brain injury (TBI) is a worldwide leading cause of mortality and long-term disability.13 TBI often causes cerebral edema, which leads to an increase in intracranial pressure (ICP) and a decrease in cerebral perfusion pressure (CPP).16,46,49 These changes result in cerebral ischemia (or hypoxia first and then eventually ischemia) and neuronal degeneration,10 which is associated with low partial brain oxygen pressure (PbtO2).33 In our previous study, we described the efficacy of 17β-estradiol (E2) in its sulfate-conjugated form (17β-estradiol sulfate, E2-SO4) to treat experimental TBI.25 E2-SO4 is a naturally occurring derivative of E2, created by the addition of a sulfate moiety via a steroid sulfotransferase enzyme. The physiological sulfate conjugation process allows excretion of this soluble form of E2 via the kidneys, which regulates the hormone levels. In our case, the solubility of E2-SO4 enables intravenous delivery of supraphysiological quantities of this hormone, which would not be possible with the highly hydrophobic native E2.

E2 is highly neuroprotective.2,9,25,29 It is synthesized in the brain with both autocrine and paracrine activities2 and freely passes the blood-brain barrier; thus, endocrine E2 is likewise at play in the CNS. While estrogen's role as a female sex hormone is well known, it is also present and synthesized normally in males, albeit at a lower level than in females. In fact, the enzyme aromatase, which is constitutive in many tissues including vertebrate brain, converts testosterone into E2.7 This process can occur in a rapid, nongenomic fashion in the brain, which supports the notion that estrogen plays a major role in brain physiology and homeostasis.40 E2 is nominally found only in neurons, but after penetrating brain injury, there is rapid de novo upregulation of aromatase in hippocampal astrocytes.12 However, E2 has a short plasma half-life (about 10 minutes), which may limit its therapeutic efficacy.

17α-ethinyl estradiol (EE) is a synthetic derivative of E2 and has a longer plasma half-life (about 10 hours) than E2.35 EE has been used for decades and has an extensive track record for efficacy and safety when used for hormone replacement therapy and birth control.1,18 However, since EE is also hydrophobic, we had 17α-ethynylestradiol-3-sulfate (EE-3-SO4) custom synthesized. EE-3-SO4 treatment in the absence of fluid resuscitation significantly increased survival of minipigs and rats that underwent severe hemorrhage (60% blood loss).17,31 These data led us to adopt EE-3-SO4 as the drug of choice for TBI treatment studies, made more relevant by a dearth of effective pharmaceuticals available to treat TBI.6

Rather than EE-3-SO4 having a lower rate of degradation or excretion, it is possible that it can be “recycled.” It has been observed that when orally administered ethinyl estradiol is conjugated and excreted, the portion that is passed in urine is completely cleared, while some of the portion entering the gut can be taken up and returned to the periphery, where the sulfate conjugate exceeds the quantity of unconjugated ethinyl estradiol.3,14 There is insufficient information on the fate of intravenously administered ethinyl estradiol (conjugated or unconjugated) to draw conclusions for the clearance and resorption, but a gut reuptake of EE-3-SO4 seems at least plausible.

TBI can also induce behavioral changes.39 Of interest, it has been reported that behavioral changes in female rats were significantly milder than those in male rats after TBI.36 This might indicate that female gonadal hormones, most likely estradiol, may alleviate sexually dimorphic stress. In fact, the improved behavioral activity of TBI models in animals was observed after estrogen treatment.5,50

In this study, we used physiological and imaging techniques to measure the salutary effect of EE-3-SO4 in a TBI rat model. The physiological measurements used were ICP, CPP, and PbtO2. The imaging methods used were diffusion tensor imaging (DTI) to assess diffuse axonal injury (DAI),4 T2 mapping to determine the size of the edematous region,24 and FDG-PET to measure brain glycolysis.11 In addition, an open-field test was performed to assess the effect of EE-3-SO4 on spontaneous exploratory behavior as a secondary injury of TBI.

Methods

Animal Study Design

Nine groups of male Sprague-Dawley rats were used (mean weight 318 ± 3 g; 10 ± 2 weeks old [± SE]); Groups 1–3 (4–5 rats per group) were used for measuring ICP, CPP, and PbtO2, Groups 4–6 (5–6 per group) were used for in vivo imaging study, and Groups 7–9 (9–11 per group) were used for open-field anxiety measurement. The groups are summarized in Table 1. Craniectomy was performed in all animals as described in our previous study.25 Groups 1, 4, and 7 were used as sham groups; in the remaining groups, TBI was induced using a lateral fluid percussion method 24 hours after completion of the craniectomy, as previously described.9 The lateral fluid percussion force was monitored and recorded from an inline transducer for quality control purposes. One hour after TBI (or 25 hours after craniectomy), each animal in Groups 3, 6, and 9 was intravenously injected with EE-3-SO4 (1 mg/kg body weight in 0.9% NaCl; 0.4 ml/kg body weight), while the other animals were intravenously injected with saline (0.9% NaCl; 0.4 ml/kg body weight). At 22 hours after dosing (or 47 hours after craniectomy), ICP, CPP, and PbtO2 levels were monitored in Groups 1–3 for 2 hours at 15-minute intervals. T2-weighted imaging, DTI, and FDG-PET/CT imaging were performed in Groups 4–6 during the same time. MRI was performed using a 9.4-T MR scanner dedicated to small animals (Bruker BioSpin Corp.), and PET/CT imaging was performed using a microPET/CT system, Triumph (GE). Animals were anesthetized using 1%–2% isoflurane during craniectomy, physiological parameter monitoring, dosing, and imaging. At 96 hours after dosing (or 121 hours after craniectomy), the open-field anxiety of animals in Groups 7–9 was assessed by measuring the duration that each animal spent in the center region of an open chamber. To reduce pain, all animals including sham animals received carprofen (intraperitoneal 5 mg/kg) and a prophylactic antibiotic (Baytril, intraperitoneal 1 mg/kg) after TBI twice per day until all experiments ended. All animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham.

TABLE 1.

Time schedule of TBI induction, dosing, ICP/PbtO2/CPP measurement, imaging or open-field measurement after craniectomy in each group

Group No.Time After Completing Craniectomy (hrs)
0242547–49121–122
1 (n = 4)CraniectomyVehicleICP/PbtO2/CPP
2 (n = 5)CraniectomyTBIVehicleICP/PbtO2/CPP
3 (n = 5)CraniectomyTBIEE-3-SO4ICP/PbtO2/CPP
4 (n = 5)CraniectomyVehicleT2/DTI/PET/CT
5 (n = 6)CraniectomyTBIVehicleT2/DTI/PET/CT
6 (n = 6)CraniectomyTBIEE-3-SO4T2/DTI/PET/CT
7 (n = 9)CraniectomyVehicleOpen-field measurement
8 (n = 11)CraniectomyTBIVehicleOpen-field measurement
9 (n = 9)CraniectomyTBIEE-3-SO4Open-field measurement

Measurement of Physiological Parameters

Physiological parameters (ICP, CPP, and PbtO2) were measured using similar methods introduced in our previous study.25 Briefly, ICP and PbtO2 were measured on the brain exposed by craniectomy using fiberoptic-based pressure transducer probes (FISO LS-10 signal conditioner and 0.9-F probe, Harvard Apparatus) and a Licox oxygen catheter microprobe (Integra Neuroscience), respectively. CPP was obtained by subtracting ICP from the mean arterial pressure, with mean arterial pressure measured using a blood pressure analyzer (Digi-Med BPA blood pressure analyzer, MicroMed Inc.) from the cannulated left femoral artery.

T2 Mapping

T2 mapping was performed to detect cerebral edema. Conventional T2-weighted images can be also used to identify the edema region, but due to nonuniform sensitivity of a surface coil over the field of view, automatic segmentation is difficult. The T2 map was obtained using the multiple TE approach.48 A multi-slice multi-echo sequence was employed for 10 1-mm-thick slices and 10 different TE values (14, 28, 42, 56, 70, 84, 98, 112, 126, and 140 msec) were used. The other detail parameters were as follows: TR 5000 msec, FOV 30 × 30 mm, and matrix size 256 × 256. The region in the ipsilateral side having T2 values larger than the mean T2 value plus 2 standard deviations of the contralateral side was determined as the edematous region, and the edema volume was calculated by the sum of all edematous regions in 10 slices. T2 values were calculated from the equation SI = Kexp(−TE/T2)t, where SI is the MR signal, K is constant, and t is time, using our own custom computer software made with MATLAB (version 7.11.0, MathWorks, Inc.). On the T2-weighted image (TE 14 msec) at the center of craniectomy, the area of the ipsilateral brain was compared with that of the contralateral side to determine whether brain morphology was changed after TBI.

Diffusion Tensor Imaging

DTI was conducted using a modified Stejskal and Tanner spin-echo diffusion-weighted sequence47 with the following imaging parameters: TR 3001 msec, TE 32 msec, FOV 30 × 30 mm, and matrix size 128 × 128. Six 1-mm-thick slices were obtained across the injured brain region. First, imaging with a b value of 0 sec/mm2 was conducted, and then diffusion gradients along 6 different directions were applied to quantitate fractional anisotropy (FA) as shown in our previous study.25 In FA maps, the region having higher FA values than the surrounding tissue was determined as the region of white matter. In each slice, the mean FA value of white matter in the ipsilateral side was divided by that in contralateral side, and the relative FA value was calculated by averaging all of these values from 6 slices. FA values were quantitated using laboratory-made computer software with MATLAB (version 7.11.0, MathWorks, Inc.).

FDG-PET/CT Imaging

The animals were not fasted prior to imaging. Animals were intravenously injected with FDG (54 ± 2 MBq in 300 μl of phosphate-buffered saline), and PET/CT imaging was applied 43 ± 1 minutes after dosing. CT imaging was conducted to identify the bone region that underwent craniectomy, as described previously.44 CT imaging was completed in 1.07 minutes, and 10 minutes of PET imaging followed. The maximum likelihood expectation maximization algorithm (10 iterations) was employed for PET image reconstruction. The field of view of each PET image slice was 46 × 46 mm, and 31 1.175-mm-thick slices were obtained. The matrix size of each PET image slice was 184 × 184. CT images were coregistered with PET images using vendor software. The animals' body temperatures were regulated to 37°C during the entire imaging process. The standardized uptake value (SUV) was calculated as (C × W)/D, where C is the tissue radioactivity per unit volume (MBq/ml), W is animal body weight (g), and D is the injected dose (MBq). In the 5 slices showing the opening for the skull, the mean SUV of the pixels in the upper half of the brain region was divided by that in the central region (about 10 mm2), and the relative SUV was determined by averaging all of these values from 5 slices. SUV was quantitated using lab-made computer software with LabVIEW (version 2010, National Instruments Co.).

Open-Field Anxiety Measurement

Open-field anxiety of animals was measured at the Behavioral Assessment Core of the University of Alabama at Birmingham. Each animal was placed at the center of a square chamber that had transparent plastic walls (width × length × height: 70 × 70 × 35 cm) and no top cover. Animal behavior was recorded using a WV-CP-484 CCD Color Surveillance camera (Panasonic) for 4 minutes. The animal track in the video was retrieved using EthoVision XT (Noldus Information Technology Inc.), a video tracking software package. The inner square (30 × 30 cm) at the center of the chamber was defined as the center region, and the duration that each animal spent in the center region was calculated. To determine whether motor and/or sensory cortex was damaged, the total distance that each animal traveled was also measured.

Statistical Analysis

SAS statistical software (version 9.4, SAS Institute Inc.) was used to conduct statistical analysis. Physiological parameters (ICP, CPP, and PbtO2) or open-field anxiety measurements among 3 groups (sham, vehicle-treated, and EE-3-SO4-treated groups) were compared using 1-way ANOVA.34 Imaging quantitated values (edema size, relative FA value, and relative SUV value) among 3 groups were compared using 1-way ANOVA as well. After Bonferroni correction for multiple comparison,34 p < 0.05 was considered statistically significant. When the p value became larger than 1 after Bonferroni correction, it was truncated to 1. In this article, the mean value is represented as the mean ± standard error.

Results

EE-3-SO4 Significantly Decreased ICP and Increased PbtO2 and CPP

Figure 1 presents the mean ICP, PbtO2, and CPP in the sham, vehicle-treated, and EE-3-SO4-treated groups, which were monitored for 2 hours (23–25 hours after dosing). The ICP of the EE-3-SO4-treated group was about 40% lower than that of the vehicle-treated group, and the difference was statistically significant for the entire 2 hours (p < 0.05). Meanwhile, PbtO2 and CPP values in the EE-3-SO4-treated group were about 60% and 10% higher than those in the vehicle-treated group, respectively; this difference was also statistically significant (p < 0.05).

FIG. 1.
FIG. 1.

Effect of EE-3-SO4 treatment on physiological parameters following TBI. ICP (A), PbtO2 (B), and CPP (C) of sham, vehicle-treated, and EE-3-SO4-treated groups at 9 different time points from 23 hours to 25 hours after administration. Asterisks and hash marks represent statistically significant differences from sham and vehicle-treated groups (4–5 per group), respectively.

EE-3-SO4 Significantly Decreased Cerebral Edema

Figure 2A shows T2-weighted images (TE 14 msec) of the brain and T2 maps of representative sham, vehicle-treated, and EE-3-SO4-treated animals after TBI in the same color scale. The ipsilateral brain regions of vehicle-and EE-3-SO4-treated animals were 4.0% ± 2.4% and 2.5% ± 1.5% larger than the contralateral brain regions, respectively, but no statistically significant difference was found between the groups (p = 0.648). Figure 2B presents the size of cerebral edema of each group. Cerebral edema was not observed in sham animals, but the mean edema size in the vehicle-treated group was 67 ± 6 mm3. The edema size of EE-3-SO4-treated animals was 25 ± 3 mm3, significantly smaller than that of vehicle-treated animals (p < 0.001).

FIG. 2.
FIG. 2.

Cerebral edema assessed by T2 mapping. A: Representative T2-weighted (T2W) images (TE 14 msec) and T2 maps of sham, vehicle-treated, and EE-3-SO4-treated animals 1 day after administration. The side that underwent craniectomy is indicated by the arrows. Edema is represented by higher T2 values (in red). B: Mean edema size in sham, vehicle-treated, and EE-3-SO4-treated groups (5–6 rats per group) at 1 day after administration. Asterisks and the hash mark represent statistically significant differences from sham and vehicle treated groups, respectively. Whiskers represent SE. Figure is available in color online only.

EE-3-SO4 Significantly Alleviated DAI

Figure 3A presents cerebral FA maps of representative sham, vehicle-treated, and EE-3-SO4-treated animals after TBI. In sham animals, the FA values of the ipsilateral white matter were not different from those of the contralateral side. However, the relative FA value of TBI-induced animals treated with vehicle was only 82.4% ± 2.2%, significantly lower than that of sham animals (p < 0.001) (Fig. 3B). When the animals were treated with EE-3-SO4, the relative FA value was increased to 90.8% ± 1.4%, which was significantly higher than that of the vehicle-treated group (p = 0.003), but lower than that of sham animals (p = 0.001).

FIG. 3.
FIG. 3.

DAI assessed by FA mapping. A: Representative FA maps of sham, vehicle-treated, and EE-3-SO4-treated animals 1 day after administration. The white matter adjacent to the craniotomy site is indicated by the arrows. B: The mean relative FA (ratio of FA value in the white matter of the ipsilateral side to that of the contralateral side) of sham, vehicle-treated, and EE-3-SO4-treated groups (5–6 rats per group) 1 day after administration. Asterisks and the hash mark represent statistically significant differences from sham and vehicle-treated groups, respectively. Whiskers represent SE.

EE-3-SO4 Significantly Increased Cerebral Glycolysis

Figure 4A presents FDG-PET/CT images of representative sham, vehicle-treated, and EE-3-SO4-treated animals after TBI, normalized to the intensity of central brain region. Figure 4B shows the relative SUV in the cerebral region of the 3 groups. The relative SUV of vehicle-treated group after TBI was 84.9% ± 1.1%, which was significantly lower than that of the sham group (100.2% ± 0.8%, p < 0.001) and the EE-3-SO4-treated group (88.9 ± 0.7%, p = 0.014). The relative SUV of EE-3-SO4-treated group, however, was significantly lower than that of the sham group (p < 0.001).

FIG. 4.
FIG. 4.

Cerebral glycolysis assessed by FDG-PET/CT imaging. A: Representative 18F-FDG PET/CT images of sham, vehicle-treated, and EE-3-SO4-treated animals 1 day after administration. The intensity of PET images is normalized to that of the central brain region. The area that underwent craniectomy is indicated by the arrows. B: The mean relative SUV (ratio of SUV averaged in the upper half of the brain to that in the central brain region) of sham, vehicle-treated, and EE-3-SO4-treated groups (5–6 rats per group) 1 day after administration. Asterisks and the hash mark represent statistically significant differences from sham and vehicle-treated groups, respectively. Whiskers represent SE. Figure is available in color online only.

EE-3-SO4 Significantly Relieved Open-Field Anxiety Induced by TBI

Figure 5A shows the representative images of the animal track. The mean distances traveled by sham, vehicle-treated, and EE-3-SO4-treated animals were 1772 ± 109, 1712 ± 117, and 1721 ± 97 cm, respectively, and no statistically significant difference was found between groups (p > 0.05). Figure 5B shows the duration that animals in each group spent in the center region. Sham animals spent 11 ± 3 seconds in the center region, whereas the vehicle-treated animals spent only 3 ± 1 seconds in the region (p = 0.018). Of note, EE-3-SO4-treated animals spent a mean of 12 ± 2 seconds in the center region, which was significantly longer than that of vehicle-treated animals (p = 0.008), and not different from that of sham animals (p = 1).

FIG. 5.
FIG. 5.

Open-field anxiety measurement. A: Representative images of the animal track in sham, vehicle-treated, and EE-3-SO4-treated groups 4 days after administration. The track is indicated by a red solid line in each subfigure. Animal movement was monitored for 4 minutes in an open box (width × length × height: 70 × 70 × 35 cm). The inner dotted square (30 × 30 cm) is defined as the center region. B: The mean duration that animals spent in the center region in each group (9–11 rats per group). The asterisk and hash mark represent statistically significant differences from sham and vehicle treated groups, respectively. Whiskers represent SE. Figure is available in color online only.

Discussion

The early salutary effects of EE-3-SO4 against TBI were confirmed using a rat model in this study. EE-3-SO4 significantly increased PbtO2, CPP, and glycolic metabolism in the brain, and decreased ICP and cerebral edema as early as 1 day after treatment compared with vehicle. These results are similar to those of estrogen sulfate (E2-SO4) in our previous study.25 Estrogen decreases vascular permeability in the injured area, increasing the transport of water molecules out of the edematous region back into circulation.30,38,45 This leads to a decrease in edema size and ICP. Estrogen also increases blood pressure during permissive hypotension,31 which may be associated with the increase of CPP and PbtO2 seen in this study. All of these factors lead to increased brain cell metabolism.

We also examined the therapeutic potential of EE-3-SO4 on behavior related to anxiety. We conducted a video tracking test of spontaneous exploratory behavior in a fenced open field, which provides a measurement of fear-related emotionality and the evolution of overall locomotor activity. The total distances that the animals traveled were not statistically different between groups, which represents that the damage to the motor and/or sensory cortex by TBI was minimal. The duration that the animals spent in the center region is associated with the ability to cope with anxiety. The EE-3-SO4-treated animals made significantly more visits to the center region than vehicle-treated animals, which indicates that EE-3-SO4 treatment decreased TBI-induced anxiety. These data are consistent with the results of a previous study, demonstrating the salutary effect of estrogen to relieve TBI-induced anxiety.36

EE-3-SO4 significantly alleviated DAI 1 day after administration; E2-SO4 was not able to do this.25 This might be related to the long plasma half-life of 17α-ethinyl estradiol (EE). EE can change its metabolism via altering enzymatic activities such as glucuronidation and sulfation in the liver,15,37,41 and it also can increase its bioavailability via interaction with specific binding proteins in the plasma.15 In addition, the binding affinity of EE to the estrogen receptors is about 2-fold higher than that of E2, which induces stronger intercellular signals.21,26,27 Furthermore, sulfated ethinyl estrogens are highly soluble in the water, which is essential for effective drug distribution in tissues.17,27,31

The rationale of DAI recovery by EE-3-SO4, however, is still not clear. We presume that EE-3-SO4 alleviates traumatic axonal injury via increasing glucose uptake to stabilize ion channels. After TBI, the intracellular pH balance and ion concentrations should be restored immediately to prevent further membrane breakdown in the neurons and axons,19 and glucose uptake is important for regulating energy-dependent ion transport proteins (especially Na/K ATPase-dependent transport proteins).52 To meet the increased needs of glucose, cerebral circulation should be enhanced.42 EE-3-SO4 can increase the cerebral circulation via its vasodilatory effects.17,31,38 Neutralizing free radicals in the damaged area using antioxidants during the early stage of injury can result in significant reduction of inflammation, edema, and axonal breakdown.51 Estrogens have strong antiinflammatory and antioxidant effects.30,45 Since EE presents higher binding affinity to estrogen receptors than natural estrogens,21,26,27 EE-3-SO4, a sulfated EE, may present higher potency against DAI, as demonstrated in this study.

In addition, a long half-life of EE-3-SO4 may effectively mitigate the secondary brain injury from TBI, reducing the likelihood of permanent brain damage and disability. TBI has primary and secondary injury phases. At the time of injury, the immediate parenchymal damages occur in the neuronal, glial, and vascular tissues, and disruptions of those cell membranes cause the disturbance of ionic homeostasis,10,46,49 which is the unavoidable primary injury. The secondary injury occurs minutes to days after the accident; cytokines and chemokines are released from immune cells after the traumatic injury, thereby promoting neuroinflammation, which triggers a cascade of physiological deterioration in tissues.8 In addition, axons within a white matter tract undergo dynamic deformations as neurons are seen to endure in both the primary and secondary injury phases, and the axonal and neuronal damages aggravate inflammation in the surrounding tissue.28,43 Antiinflammatory effects of estrogens are well established,45 and therefore EE-3-SO4 may be able to successfully prevent the secondary injury as it presents increased efficacy and gives longer exposure. EE-3-SO4 may also be able to prevent cerebral vasospasm and edema that occur during the secondary injury phase.

It should be noted that serious injuries on the battlefield and in civilian settings are often polytraumatic, and thus TBI may present simultaneously with hemorrhage. This offers the intriguing prospect that pharmacological estrogen treatment could serve a dual purpose. Since hemorrhage is obvious, our proposed treatment could be given immediately for the treatment of hemorrhage and in lieu of a TBI diagnosis, where TBI may or may not be obvious. In so doing, both TBI and hemorrhage would benefit from exposure to estrogen as early as possible, preferably within the “golden hour.” In addition, previous work supports the use of estrogen as prophylaxis or treatment for sepsis,22,23 which is another likely component of polytrauma, especially in cases of penetrating wounds. Also, it should be noted that plans are under way to conduct clinical trials with EE-3-SO4 for treating severe hemorrhage, beginning with the DARPA (Defense Advanced Research Agency) Surviving Blood Loss initiative, which specified that large and small animals be treated with a drug without immediate fluid resuscitation. In those studies,18,34 we were able to demonstrate a 6-hour survival with no interventions other than EE-3-SO4 administration in rats and pigs, delivered in a very small, nonresuscitative volume (0.4 ml/kg body weight).

One additional prospect for administration of exogenous estrogen is a secondary or “knock on” effect. An interesting example in this context is the stimulation of the pituitary to secrete prolactin by exogenous estrogen administration, which has been demonstrated in healthy women,3 as well as with cultured pituitary cells.20 It has been found that prolactin is also neuroprotective, where it minimizes the damage from excitotoxicity in the hippocampus. This has been modeled in rats with kainic acid, which induces seizures. The investigators observed that prolactin greatly reduces the number and progression of seizures.32

Although our study has shown salutary effects of EE-3-SO4 treatment after TBI on various parameters including edema reduction, it should be noted that the earliest measurements were carried out 23 hours posttreatment. Thus, it remains unknown if the salutary effects are observed even earlier than 23 hours after treatment. Likewise, it remains unknown if the salutary effects persist longer than the time frame in this study. It would appear, however, that the salutary effects of EE-3-SO4 do persist after 24 hours since these parameters do normalize after a week or so in this model of mild to moderate TBI, which is supported by the results of the open-field anxiety test performed 4 days after TBI in this study. Thus, it appears that the use of EE-3-SO4 after TBI accelerates the recovery of all the measured parameters. It should also be noted that only a single dose of EE-3-SO4 (i.e., 1 mg/kg body weight) was used in this study. Thus, it remains unknown if a higher dose of EE-3-SO4 would be even more efficacious in faster recovery of the altered parameters to normal.

Conclusions

EE-3-SO4 as a drug will be stable, highly soluble, and have minimal space requirements, which will facilitate easy transport of the agent for military medics and civilian first responders. An intraosseous autoinjector will allow for battlefield self- or “buddy” administration as well. Our hope is that this drug will prove to be lifesaving and may prevent long-term disability from TBI. However, as our study was based on a single TBI rodent model and histological analyses were not implemented, more extensive preclinical studies and clinical trials will need to follow to confirm the utility of EE-3-SO4 in battlefield or with civilian trauma settings.

Acknowledgments

We thank Dr. Ahmar Ayub for inducing TBI in the rats and Dr. Guihua Zhai and Sharon Samuel for data collection and analyses. This study was supported by the US Army Medical Research and Material Command, Office of Congressional Directed Medical Research Program in W81XWH-08-2-0153 of the Department of Defense to I.H.C.

Disclosures

The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

Author Contributions

Conception and design: Chaudry, Kim, Yu, van Groen, Hubbard. Acquisition of data: Kim, Yu, Cam-Etoz. Analysis and interpretation of data: all authors. Drafting the article: Kim, Yu, Cam-Etoz, Hubbard. Critically revising the article: all authors. Reviewed submitted version of manuscript: all authors. Approved the final version of the manuscript on behalf of all authors: Chaudry. Statistical analysis: Kim. Administrative/technical/material support: Chaudry, Kim, Yu, van Groen, Hubbard. Study supervision: Chaudry, Kim, van Groen, Hubbard.

Supplemental Information

Previous Presentations

A portion of the contents of this paper was presented at World Molecular Imaging Congress in Savannah, Georgia, September 18–21, 2013.

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    Abrams LSSkee DMNatarajan JWong FALeese PTCreasy GW: Pharmacokinetics of norelgestromin and ethinyl estradiol delivered by a contraceptive patch (Ortho Evra/Evra) under conditions of heat, humidity, and exercise. J Clin Pharmacol 41:130113092001

  • 2

    Arevalo MAAzcoitia IGarcia-Segura LM: The neuroprotective actions of oestradiol and oestrogen receptors. Nat Rev Neurosci 16:17292015

  • 3

    Back DJBolt HMBreckenridge AMCrawford FEOrme MLRowe PH: The pharmacokinetics of a large (3 mg) oral dose of ethynylestradiol in women. Contraception 21:1451531980

  • 4

    Bodanapally UKShanmuganathan KSaksobhavivat NSliker CWMiller LAChoi AY: MR imaging and differentiation of cerebral fat embolism syndrome from diffuse axonal injury: application of diffusion tensor imaging. Neuroradiology 55:7717782013

  • 5

    Bowman REFerguson DLuine VN: Effects of chronic restraint stress and estradiol on open field activity, spatial memory, and monoaminergic neurotransmitters in ovariectomized rats. Neuroscience 113:4014102002

  • 6

    Carpenter KLCzosnyka MJalloh INewcombe VFHelmy AShannon RJ: Systemic, local, and imaging biomarkers of brain injury: more needed, and better use of those already established?. Front Neurol 6:262015

  • 7

    Charlier TDCornil CABalthazart J: Rapid modulation of aromatase activity in the vertebrate brain. J Exp Neurosci 7:31372013

  • 8

    Corps KNRoth TLMcGavern DB: Inflammation and neuroprotection in traumatic brain injury. JAMA Neurol 72:3553622015

  • 9

    Day NLFloyd CLD'Alessandro TLHubbard WJChaudry IH: 17β-estradiol confers protection after traumatic brain injury in the rat and involves activation of G protein-coupled estrogen receptor 1. J Neurotrauma 30:153115412013

  • 10

    DeWitt DSJenkins LWPrough DS: Enhanced vulnerability to secondary ischemic insults after experimental traumatic brain injury. New Horiz 3:3763831995

  • 11

    García-Panach JLull NLull JJFerri JMartínez CSopena P: A voxel-based analysis of FDG-PET in traumatic brain injury: regional metabolism and relationship between the thalamus and cortical areas. J Neurotrauma 28:170717172011

  • 12

    Garcia-Segura LMWozniak AAzcoitia IRodriguez JRHutchison REHutchison JB: Aromatase expression by astrocytes after brain injury: implications for local estrogen formation in brain repair. Neuroscience 89:5675781999

  • 13

    Gean ADFischbein NJ: Head trauma. Neuroimaging Clin N Am 20:5275562010

  • 14

    Goldzieher JWBrody SA: Pharmacokinetics of ethinyl estradiol and mestranol. Am J Obstet Gynecol 163:211421191990

  • 15

    Guengerich FP: Inhibition of oral contraceptive steroid-metabolizing enzymes by steroids and drugs. Am J Obstet Gynecol 163:215921631990

  • 16

    Hiler MCzosnyka MHutchinson PBalestreri MSmielewski PMatta B: Predictive value of initial computerized tomography scan, intracranial pressure, and state of autoregulation in patients with traumatic brain injury. J Neurosurg 104:7317372006

  • 17

    Hubbard WKeith JBerman JMiller MScott CPeck C: 17α-Ethynylestradiol-3-sulfate treatment of severe blood loss in rats. J Surg Res 193:3553602015

  • 18

    Jick SSKaye JARussmann SJick H: Risk of nonfatal venous thromboembolism in women using a contraceptive transdermal patch and oral contraceptives containing norgestimate and 35 microg of ethinyl estradiol. Contraception 73:2232282006

  • 19

    Johnson VEStewart WSmith DH: Axonal pathology in traumatic brain injury. Exp Neurol 246:35432013

  • 20

    Jones GEBoyns AR: Oestradiol stimulation of prolactin release from canine pituitary in culture. Acta Endocrinol (Copenh) 82:7067091976

  • 21

    Jung-Hoffmann CFitzner MKuhl H: Oral contraceptives containing 20 or 30 micrograms ethinylestradiol and 150 micrograms desogestrel: pharmacokinetics and pharmacodynamic parameters. Horm Res 36:2382461991

  • 22

    Kawasaki TChaudry IH: The effects of estrogen on various organs: therapeutic approach for sepsis, trauma, and reperfusion injury. Part 1: central nervous system, lung, and heart. J Anesth 26:8838912012

  • 23

    Kawasaki TChaudry IH: The effects of estrogen on various organs: therapeutic approach for sepsis, trauma, and reperfusion injury. Part 2: liver, intestine, spleen, and kidney. J Anesth 26:8928992012

  • 24

    Kharatishvili ISierra AImmonen RJGröhn OHPitkänen A: Quantitative T2 mapping as a potential marker for the initial assessment of the severity of damage after traumatic brain injury in rat. Exp Neurol 217:1541642009

  • 25

    Kim HCam-Etoz BZhai GHubbard WJZinn KRChaudry IH: Salutary effects of estrogen sulfate for traumatic brain injury. J Neurotrauma 32:121012162015

  • 26

    Lindberg UBCrona NStigendal LTeger-Nilsson ACSilfverstolpe G: A comparison between effects of estradiol valerate and low dose ethinyl estradiol on haemostasis parameters. Thromb Haemost 61:65691989

  • 27

    Lindberg UBEnk LCrona NSilfverstolpe G: A comparison of the effects of ethinyl estradiol and estradiol valerate on serum and lipoprotein lipids. Maturitas 10:3433521988

  • 28

    Liu YRCardamone LHogan REGregoire MCWilliams JPHicks RJ: Progressive metabolic and structural cerebral perturbations after traumatic brain injury: an in vivo imaging study in the rat. J Nucl Med 51:178817952010

  • 29

    Manthey DBehl C: From structural biochemistry to expression profiling: neuroprotective activities of estrogen. Neuroscience 138:8458502006

  • 30

    McEwen B: Estrogen actions throughout the brain. Recent Prog Horm Res 57:3573842002

  • 31

    Miller MKeith JBerman JBurlington DBGrudzinskas CHubbard W: Efficacy of 17α-ethynylestradiol-3-sulfate for severe hemorrhage in minipigs in the absence of fluid resuscitation. J Trauma Acute Care Surg 76:140914162014

  • 32

    Morales T: Recent findings on neuroprotection against excitotoxicity in the hippocampus of female rats. J Neuroendocrinol 23:99410012011

  • 33

    Murakami YWei GYang XLu XCLeung LYShear DA: Brain oxygen tension monitoring following penetrating ballistic-like brain injury in rats. J Neurosci Methods 203:1151212012

  • 34

    Neter JKutner MHNachtsheim JCWasserman W: Applied Linear Statistical Models ed 4ColumbusMcGraw-Hill1996

  • 35

    Obach RSLombardo FWaters NJ: Trend analysis of a database of intravenous pharmacokinetic parameters in humans for 670 drug compounds. Drug Metab Dispos 36:138514052008

  • 36

    O'Connor CACernak IVink R: Interaction between anesthesia, gender, and functional outcome task following diffuse traumatic brain injury in rats. J Neurotrauma 20:5335412003

  • 37

    Ortiz de Montellano PRKunze KL: Self-catalyzed inactivation of hepatic cytochrome P-450 by ethynyl substrates. J Biol Chem 255:557855851980

  • 38

    Roof RLHall ED: Estrogen-related gender difference in survival rate and cortical blood flow after impact-acceleration head injury in rats. J Neurotrauma 17:115511692000

  • 39

    Ryan NPCatroppa CBeare RSilk TJCrossley LBeauchamp MH: Theory of mind mediates the prospective relationship between abnormal social brain network morphology and chronic behavior problems after pediatric traumatic brain injury. Soc Cogn Affect Neurosci 11:6836922016

  • 40

    Santollo JDaniels D: Control of fluid intake by estrogens in the female rat: role of the hypothalamus. Front Syst Neurosci 9:252015

  • 41

    Schindler AE: Noncontraceptive benefits of oral hormonal contraceptives. Int J Endocrinol Metab 11:41472013

  • 42

    Selwyn RHockenbury NJaiswal SMathur SArmstrong RCByrnes KR: Mild traumatic brain injury results in depressed cerebral glucose uptake: An 18FDG PET study. J Neurotrauma 30:194319532013

  • 43

    Seo JPKim OLKim SHChang MCKim MSSon SM: Neural injury of uncinate fasciculus in patients with diffuse axonal injury. NeuroRehabilitation 30:3233282012

  • 44

    Shah NZhai GKnowles JAStockard CRGrizzle WEFineberg N: 18F-FDG PET/CT imaging detects therapy efficacy of anti-EMMPRIN antibody and gemcitabine in orthotopic pancreatic tumor xenografts. Mol Imaging Biol 14:2372442012

  • 45

    Stein DGHoffman SW: Estrogen and progesterone as neuroprotective agents in the treatment of acute brain injuries. Pediatr Rehabil 6:13222003

  • 46

    Stocchetti NColombo AOrtolano FVidetta WMarchesi RLonghi L: Time course of intracranial hypertension after traumatic brain injury. J Neurotrauma 24:133913462007

  • 47

    Tofts PS: Quantitative MRI of the Brain ChichesterWiley2003

  • 48

    Tofts PSdu Boulay EP: Towards quantitative measurements of relaxation times and other parameters in the brain. Neuroradiology 32:4074151990

  • 49

    Unterberg AWStover JKress BKiening KL: Edema and brain trauma. Neuroscience 129:102110292004

  • 50

    Walf AAFrye CA: A review and update of mechanisms of estrogen in the hippocampus and amygdala for anxiety and depression behavior. Neuropsychopharmacology 31:109711112006

  • 51

    Wang GHJiang ZLLi YCLi XShi HGao YQ: Free-radical scavenger edaravone treatment confers neuroprotection against traumatic brain injury in rats. J Neurotrauma 28:212321342011

  • 52

    Yuen TJBrowne KDIwata ASmith DH: Sodium channelopathy induced by mild axonal trauma worsens outcome after a repeat injury. J Neurosci Res 87:362036252009

If the inline PDF is not rendering correctly, you can download the PDF file here.

Article Information

Correspondence Irshad H. Chaudry, Department of Surgery, University of Alabama at Birmingham, VH G094, 1670 University Blvd., Birmingham, AL 35294-0019. email: ichaudry@uab.edu.

INCLUDE WHEN CITING Published online September 23, 2016; DOI: 10.3171/2016.7.JNS161263.

Disclosures The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

© AANS, except where prohibited by US copyright law.

Headings

Figures

  • View in gallery

    Effect of EE-3-SO4 treatment on physiological parameters following TBI. ICP (A), PbtO2 (B), and CPP (C) of sham, vehicle-treated, and EE-3-SO4-treated groups at 9 different time points from 23 hours to 25 hours after administration. Asterisks and hash marks represent statistically significant differences from sham and vehicle-treated groups (4–5 per group), respectively.

  • View in gallery

    Cerebral edema assessed by T2 mapping. A: Representative T2-weighted (T2W) images (TE 14 msec) and T2 maps of sham, vehicle-treated, and EE-3-SO4-treated animals 1 day after administration. The side that underwent craniectomy is indicated by the arrows. Edema is represented by higher T2 values (in red). B: Mean edema size in sham, vehicle-treated, and EE-3-SO4-treated groups (5–6 rats per group) at 1 day after administration. Asterisks and the hash mark represent statistically significant differences from sham and vehicle treated groups, respectively. Whiskers represent SE. Figure is available in color online only.

  • View in gallery

    DAI assessed by FA mapping. A: Representative FA maps of sham, vehicle-treated, and EE-3-SO4-treated animals 1 day after administration. The white matter adjacent to the craniotomy site is indicated by the arrows. B: The mean relative FA (ratio of FA value in the white matter of the ipsilateral side to that of the contralateral side) of sham, vehicle-treated, and EE-3-SO4-treated groups (5–6 rats per group) 1 day after administration. Asterisks and the hash mark represent statistically significant differences from sham and vehicle-treated groups, respectively. Whiskers represent SE.

  • View in gallery

    Cerebral glycolysis assessed by FDG-PET/CT imaging. A: Representative 18F-FDG PET/CT images of sham, vehicle-treated, and EE-3-SO4-treated animals 1 day after administration. The intensity of PET images is normalized to that of the central brain region. The area that underwent craniectomy is indicated by the arrows. B: The mean relative SUV (ratio of SUV averaged in the upper half of the brain to that in the central brain region) of sham, vehicle-treated, and EE-3-SO4-treated groups (5–6 rats per group) 1 day after administration. Asterisks and the hash mark represent statistically significant differences from sham and vehicle-treated groups, respectively. Whiskers represent SE. Figure is available in color online only.

  • View in gallery

    Open-field anxiety measurement. A: Representative images of the animal track in sham, vehicle-treated, and EE-3-SO4-treated groups 4 days after administration. The track is indicated by a red solid line in each subfigure. Animal movement was monitored for 4 minutes in an open box (width × length × height: 70 × 70 × 35 cm). The inner dotted square (30 × 30 cm) is defined as the center region. B: The mean duration that animals spent in the center region in each group (9–11 rats per group). The asterisk and hash mark represent statistically significant differences from sham and vehicle treated groups, respectively. Whiskers represent SE. Figure is available in color online only.

References

1

Abrams LSSkee DMNatarajan JWong FALeese PTCreasy GW: Pharmacokinetics of norelgestromin and ethinyl estradiol delivered by a contraceptive patch (Ortho Evra/Evra) under conditions of heat, humidity, and exercise. J Clin Pharmacol 41:130113092001

2

Arevalo MAAzcoitia IGarcia-Segura LM: The neuroprotective actions of oestradiol and oestrogen receptors. Nat Rev Neurosci 16:17292015

3

Back DJBolt HMBreckenridge AMCrawford FEOrme MLRowe PH: The pharmacokinetics of a large (3 mg) oral dose of ethynylestradiol in women. Contraception 21:1451531980

4

Bodanapally UKShanmuganathan KSaksobhavivat NSliker CWMiller LAChoi AY: MR imaging and differentiation of cerebral fat embolism syndrome from diffuse axonal injury: application of diffusion tensor imaging. Neuroradiology 55:7717782013

5

Bowman REFerguson DLuine VN: Effects of chronic restraint stress and estradiol on open field activity, spatial memory, and monoaminergic neurotransmitters in ovariectomized rats. Neuroscience 113:4014102002

6

Carpenter KLCzosnyka MJalloh INewcombe VFHelmy AShannon RJ: Systemic, local, and imaging biomarkers of brain injury: more needed, and better use of those already established?. Front Neurol 6:262015

7

Charlier TDCornil CABalthazart J: Rapid modulation of aromatase activity in the vertebrate brain. J Exp Neurosci 7:31372013

8

Corps KNRoth TLMcGavern DB: Inflammation and neuroprotection in traumatic brain injury. JAMA Neurol 72:3553622015

9

Day NLFloyd CLD'Alessandro TLHubbard WJChaudry IH: 17β-estradiol confers protection after traumatic brain injury in the rat and involves activation of G protein-coupled estrogen receptor 1. J Neurotrauma 30:153115412013

10

DeWitt DSJenkins LWPrough DS: Enhanced vulnerability to secondary ischemic insults after experimental traumatic brain injury. New Horiz 3:3763831995

11

García-Panach JLull NLull JJFerri JMartínez CSopena P: A voxel-based analysis of FDG-PET in traumatic brain injury: regional metabolism and relationship between the thalamus and cortical areas. J Neurotrauma 28:170717172011

12

Garcia-Segura LMWozniak AAzcoitia IRodriguez JRHutchison REHutchison JB: Aromatase expression by astrocytes after brain injury: implications for local estrogen formation in brain repair. Neuroscience 89:5675781999

13

Gean ADFischbein NJ: Head trauma. Neuroimaging Clin N Am 20:5275562010

14

Goldzieher JWBrody SA: Pharmacokinetics of ethinyl estradiol and mestranol. Am J Obstet Gynecol 163:211421191990

15

Guengerich FP: Inhibition of oral contraceptive steroid-metabolizing enzymes by steroids and drugs. Am J Obstet Gynecol 163:215921631990

16

Hiler MCzosnyka MHutchinson PBalestreri MSmielewski PMatta B: Predictive value of initial computerized tomography scan, intracranial pressure, and state of autoregulation in patients with traumatic brain injury. J Neurosurg 104:7317372006

17

Hubbard WKeith JBerman JMiller MScott CPeck C: 17α-Ethynylestradiol-3-sulfate treatment of severe blood loss in rats. J Surg Res 193:3553602015

18

Jick SSKaye JARussmann SJick H: Risk of nonfatal venous thromboembolism in women using a contraceptive transdermal patch and oral contraceptives containing norgestimate and 35 microg of ethinyl estradiol. Contraception 73:2232282006

19

Johnson VEStewart WSmith DH: Axonal pathology in traumatic brain injury. Exp Neurol 246:35432013

20

Jones GEBoyns AR: Oestradiol stimulation of prolactin release from canine pituitary in culture. Acta Endocrinol (Copenh) 82:7067091976

21

Jung-Hoffmann CFitzner MKuhl H: Oral contraceptives containing 20 or 30 micrograms ethinylestradiol and 150 micrograms desogestrel: pharmacokinetics and pharmacodynamic parameters. Horm Res 36:2382461991

22

Kawasaki TChaudry IH: The effects of estrogen on various organs: therapeutic approach for sepsis, trauma, and reperfusion injury. Part 1: central nervous system, lung, and heart. J Anesth 26:8838912012

23

Kawasaki TChaudry IH: The effects of estrogen on various organs: therapeutic approach for sepsis, trauma, and reperfusion injury. Part 2: liver, intestine, spleen, and kidney. J Anesth 26:8928992012

24

Kharatishvili ISierra AImmonen RJGröhn OHPitkänen A: Quantitative T2 mapping as a potential marker for the initial assessment of the severity of damage after traumatic brain injury in rat. Exp Neurol 217:1541642009

25

Kim HCam-Etoz BZhai GHubbard WJZinn KRChaudry IH: Salutary effects of estrogen sulfate for traumatic brain injury. J Neurotrauma 32:121012162015

26

Lindberg UBCrona NStigendal LTeger-Nilsson ACSilfverstolpe G: A comparison between effects of estradiol valerate and low dose ethinyl estradiol on haemostasis parameters. Thromb Haemost 61:65691989

27

Lindberg UBEnk LCrona NSilfverstolpe G: A comparison of the effects of ethinyl estradiol and estradiol valerate on serum and lipoprotein lipids. Maturitas 10:3433521988

28

Liu YRCardamone LHogan REGregoire MCWilliams JPHicks RJ: Progressive metabolic and structural cerebral perturbations after traumatic brain injury: an in vivo imaging study in the rat. J Nucl Med 51:178817952010

29

Manthey DBehl C: From structural biochemistry to expression profiling: neuroprotective activities of estrogen. Neuroscience 138:8458502006

30

McEwen B: Estrogen actions throughout the brain. Recent Prog Horm Res 57:3573842002

31

Miller MKeith JBerman JBurlington DBGrudzinskas CHubbard W: Efficacy of 17α-ethynylestradiol-3-sulfate for severe hemorrhage in minipigs in the absence of fluid resuscitation. J Trauma Acute Care Surg 76:140914162014

32

Morales T: Recent findings on neuroprotection against excitotoxicity in the hippocampus of female rats. J Neuroendocrinol 23:99410012011

33

Murakami YWei GYang XLu XCLeung LYShear DA: Brain oxygen tension monitoring following penetrating ballistic-like brain injury in rats. J Neurosci Methods 203:1151212012

34

Neter JKutner MHNachtsheim JCWasserman W: Applied Linear Statistical Models ed 4ColumbusMcGraw-Hill1996

35

Obach RSLombardo FWaters NJ: Trend analysis of a database of intravenous pharmacokinetic parameters in humans for 670 drug compounds. Drug Metab Dispos 36:138514052008

36

O'Connor CACernak IVink R: Interaction between anesthesia, gender, and functional outcome task following diffuse traumatic brain injury in rats. J Neurotrauma 20:5335412003

37

Ortiz de Montellano PRKunze KL: Self-catalyzed inactivation of hepatic cytochrome P-450 by ethynyl substrates. J Biol Chem 255:557855851980

38

Roof RLHall ED: Estrogen-related gender difference in survival rate and cortical blood flow after impact-acceleration head injury in rats. J Neurotrauma 17:115511692000

39

Ryan NPCatroppa CBeare RSilk TJCrossley LBeauchamp MH: Theory of mind mediates the prospective relationship between abnormal social brain network morphology and chronic behavior problems after pediatric traumatic brain injury. Soc Cogn Affect Neurosci 11:6836922016

40

Santollo JDaniels D: Control of fluid intake by estrogens in the female rat: role of the hypothalamus. Front Syst Neurosci 9:252015

41

Schindler AE: Noncontraceptive benefits of oral hormonal contraceptives. Int J Endocrinol Metab 11:41472013

42

Selwyn RHockenbury NJaiswal SMathur SArmstrong RCByrnes KR: Mild traumatic brain injury results in depressed cerebral glucose uptake: An 18FDG PET study. J Neurotrauma 30:194319532013

43

Seo JPKim OLKim SHChang MCKim MSSon SM: Neural injury of uncinate fasciculus in patients with diffuse axonal injury. NeuroRehabilitation 30:3233282012

44

Shah NZhai GKnowles JAStockard CRGrizzle WEFineberg N: 18F-FDG PET/CT imaging detects therapy efficacy of anti-EMMPRIN antibody and gemcitabine in orthotopic pancreatic tumor xenografts. Mol Imaging Biol 14:2372442012

45

Stein DGHoffman SW: Estrogen and progesterone as neuroprotective agents in the treatment of acute brain injuries. Pediatr Rehabil 6:13222003

46

Stocchetti NColombo AOrtolano FVidetta WMarchesi RLonghi L: Time course of intracranial hypertension after traumatic brain injury. J Neurotrauma 24:133913462007

47

Tofts PS: Quantitative MRI of the Brain ChichesterWiley2003

48

Tofts PSdu Boulay EP: Towards quantitative measurements of relaxation times and other parameters in the brain. Neuroradiology 32:4074151990

49

Unterberg AWStover JKress BKiening KL: Edema and brain trauma. Neuroscience 129:102110292004

50

Walf AAFrye CA: A review and update of mechanisms of estrogen in the hippocampus and amygdala for anxiety and depression behavior. Neuropsychopharmacology 31:109711112006

51

Wang GHJiang ZLLi YCLi XShi HGao YQ: Free-radical scavenger edaravone treatment confers neuroprotection against traumatic brain injury in rats. J Neurotrauma 28:212321342011

52

Yuen TJBrowne KDIwata ASmith DH: Sodium channelopathy induced by mild axonal trauma worsens outcome after a repeat injury. J Neurosci Res 87:362036252009

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