Background
Traumatic brain injury (TBI) and severe blood loss resulting in hemorrhagic shock (HS) individually represent leading causes of trauma-induced mortality and are especially detrimental when combined [
1‐
3]. Hypothermia, acidosis and coagulopathy represent the hallmark complications of HS, ultimately resulting in oxygen debt at the tissue level [
4]. Hypovolemia also decreases arterial pressure and increases vasoconstriction, resulting in earlier and more severe cerebral dysautoregulation, reduced blood flow, hypoxia, increased contusion volume and a doubling of mortality rate in concurrent TBI + HS [
5,
6]. Although fluid resuscitation is recommended for HS, unless carefully managed it can also exacerbate brain edema and elevate intracranial pressure [
7]. Similarly, hyperventilation helps to restore systemic acid–base balance following HS [
8], whereas respiratory depression is the most common cause of death in preclinical models of moderate-to-severe TBI [
9]. Respiratory complications are also more common in TBI + HS relative to HS alone in humans [
10]. Thus, the optimal resuscitation approaches for concurrent TBI + HS remain actively debated [
3,
7].
Care is further complicated in remote settings (e.g., injuries occurring in the wilderness, developing countries, or military settings) where resuscitative fluids and blood products are not readily available [
11,
12]. Death typically ensues in little more than one hour in the absence of intravenous fluid resuscitation in Class III or IV trauma patients [
13]. The family of estrogens (17β-estradiol and 17α-ethinyl estradiol-3-sulfate [EE-3-SO
4]) are naturally occurring steroid hormones that are beneficial in HS [
14,
15] and have been shown to be neuroprotective across multiple neural injury models [
16,
17]. Specifically, EE-3-SO
4 has been shown to increase short-term survival rate (e.g., 3–6 h) in both rodent [
18] and swine [
15] models of HS in the absence of typical doses of resuscitation fluids. Proposed mechanisms of action for EE-3-SO
4 include increased cardiac ejection fraction and vasodilation [
19‐
22], increased mitochondrial respiratory complex activity in the myocardium [
18,
23], increased cell survival pathways concomitant with decreased cell death pathways, as well as decreased metabolic acidosis and glucose derangement [
15,
21]. All of the above effects are dependent on estrogen receptor engagement, where specificity was recently confirmed by estrogen receptor antagonists [
20].
Estrogens freely cross the blood–brain barrier (BBB) and have been demonstrated to maintain and regulate the BBB in both humans and rodents [
24,
25]. In terms of neuroprotection, seminal work suggests estrogens reduce lesion size and lessen the extent of cell death in the injured brain [
17], as well as potentially promoting vascular regeneration following injury [
26]. It has been suggested that the estrogen-mediated maintenance of the BBB may also reduce edema after stroke via dampening of the Na–K–Cl cotransporter mechanism [
27]. Most pertinent to the current study, EE-3-SO
4 administered one hour following TBI in rodent models resulted in a reduction in intracranial pressure, edema and neuroinflammation while increasing cerebral perfusion pressure and partial pressure of oxygen in brain tissue [
17,
28‐
30], but did not affect markers of diffuse axonal injury [
30].
Due to similar homology in terms of hemostatic mechanisms, cardiovascular systems and brain structure (gyrencephalic, similar gray-white matter ratios), swine represent the most commonly utilized species for large animal models of TBI + HS [
3]. The majority of these studies have primarily utilized controlled cortical impact or fluid percussion injury, even though closed-head, acceleration injuries represent the most common form of human TBI [
31]. A recent study reported acute mortality rates of approximately 88% and 13%, respectively, in an acceleration model of TBI with either 55% or 40% blood loss in the absence of any treatment relative to Shams [
32]. In addition to traditional metrics of metabolic derangement associated with HS, results from this study also validated the sensitivity of several blood-based biomarkers for measuring diffuse axonal injury, blood–brain barrier breach and neuroinflammation in swine (glial fibrillary acidic protein [GFAP], neurofilament light chain [NFL], ubiquitin C-terminal hydrolase [UCH-L1], amyloid-beta 40 [Aβ40] and 42 [Aβ42]) that are commonly used in clinical settings [
33]. To our knowledge, there have been no studies examining the efficacy of EE-3-SO
4 in a large animal model of TBI + HS.
The current study therefore had two primary aims. The first was to attempt to replicate previous findings of metabolic derangement and neurotrauma in a swine model of closed-head, accelerative TBI + HS (i.e., Placebo-treated animals relative to uninjured Shams). The second aim examined the efficacy of EE-3-SO
4 to prolong survival in a pre-hospital environment that mimicked more austere levels of care (absence of resuscitative fluids or mechanical ventilation; [
32]). Based on previous literature [
15,
21], we postulated that EE-3-SO
4 would increase survival time and improve hemodynamic functioning, while subsequently decreasing markers of metabolic acidosis, BBB breach and neuroinflammation. Second, we also predicted that there would be a statistically null effect for blood-based and immunohistochemical markers of diffuse axonal injury [
30].
Discussion
Blood products are not always available in extreme circumstances [
1,
36], necessitating the development of novel agents that can both augment the body’s natural response to severe blood loss and mitigate pathological aspects of shock [
4]. The current study examined the efficacy of EE-3-SO
4 as a treatment for TBI + HS in an austere environment (no mechanical ventilation post-injury, no additional resuscitation fluids, no craniotomy), as frequently occurs in military trauma scenarios and in developing countries [
32]. The blocked randomization procedures adequately controlled for all potential major confounders from both demographic variables (non-significant differences in animal age, weight and sex) and experimental (statistically equivalent TBI exposure parameters, pre-injury anesthetic time, blood loss levels, etc.) procedures. Current results replicated previous findings of metabolic derangements, a decrease in MAP in conjunction with increased heart-rate, and both blood-based and immunohistochemical evidence of diffuse axonal injury and blood brain barrier disruption in a large animal model of closed-head accelerative TBI + HS [
32].
The administration of EE-3-SO
4 increased survival rate, normalized pulse pressure immediately post-drug, and provided preliminary evidence of neuroprotection relative to the Placebo cohort in this fully blinded trial. Previous studies have demonstrated increased survival rates and times for rodent and swine models of HS following intravenous EE-3-SO
4 administration [
15,
21]. Current findings extend these results to a large animal model of TBI + HS with approximately 40% blood loss, with EE-3-SO
4 significantly prolonging survival rate relative to a control cohort (90.3% vs. 72.7%, respectively), albeit at a smaller magnitude relative to previous studies of isolated and severe HS [
15]. The mortality rate observed in the Placebo cohort was also roughly commensurate with reported Class III–IV trauma rates [
13], providing additional external validity for the closed-head TBI + HS model.
The majority of animals in both groups expired from respiratory distress rather than cardiovascular factors, similar to a previous TBI + HS swine model with 55% blood loss [
32]. Acute respiratory failure represents the leading cause of death in preclinical models of isolated TBI [
9], complicates clinical care of TBI patients [
37], and is more common following TBI + HS relative to HS alone [
10]. The current study did not directly quantify the presence of congestion, edema, hemorrhage or microatelectasis in pulmonary tissue as has been done in previous swine models [
38], complicating the dissociation of central nervous system involvement in respiratory failure due to TBI. Mechanical ventilation remains the first line of defense for managing acute respiratory distress syndrome in both pre-hospital and hospital setting following complex trauma [
39] and is typically used during all phases of preclinical trauma models [
3]. However, mechanical ventilation is not available as a treatment option in austere environments to combat respiratory distress, representing a potentially critical factor that should be more carefully considered in future studies for full bench-to-bedside translation.
EE-3-SO
4 also more rapidly restored pulse pressure post-administration relative to Placebo, followed by statistically equivalent pressures for the remainder of the experiment. The rapid action of EE-3-SO
4 on pulse pressure suggests direct activation of estrogen receptors rather than through genomic signaling [
20]. The membrane receptor effects of estrogen include activation of endothelial nitric oxide synthase and the consequent production of nitric oxide, as well as endothelial-independent, rapid mobilization and release of calcium within subcellular compartments leading to increases in Caþþ-triggered Kþ channel activity [
22]. Activation of these receptors collectively results in changes to myocardial contractility and vasodilation of vascular smooth muscle [
20]. Over-exuberant vasodilation in the face of severe hypovolemia could be detrimental, but initial vasodilation could also moderate the intense peripheral vasoconstriction seen in TBI + HS, and contribute to the normalization of pulse pressure.
Replication analyses indicated significant post-injury changes in all point-of-care markers of acidosis and other metabolic derangements (glucose, lactate, bicarbonate, etc.), the majority of which did not recover to baseline levels at the end of the 5 h monitoring period. Several of point-of-care markers (pH and potassium) were significantly more affected in non-surviving relative to surviving animals
prior to death, although glucose was unexpectedly higher for surviving animals. Acidosis represents one of the hallmark complications of HS, and non-surviving animals were unable to compensate from a hemodynamic perspective, ultimately resulting in even further increases in oxygen debt at the tissue level [
4,
40]. With the exception of glucose, current findings also partially replicate a previous swine model of severe hemorrhage, which reported that increased glucose/potassium/lactate and decreased bicarbonate/MAP were associated with survival [
15]. In contrast to previous work in rodents [
20], EE-3-SO
4 administration did not significantly affect either point-of-care markers or MAP relative to Placebo, suggesting the need for polytherapeutic approaches to further promote survival and more rapidly restore homeostasis following TBI + HS [
7].
Clinical research studies are increasingly using blood-based protein assays to characterize the extent of neurotrauma both in the acute and chronic injury phases of TBI [
33]. Previous findings [
32] of significant changes in NFL, GFAP and Aβ42 were replicated in our swine models of accelerative TBI + HS, with UCH-L1 and Aβ40 also significant in the current study due to increased statistical power. Several of these blood-based biomarkers demonstrated sensitivity to injury as soon as 35 min post-TBI and were strongly associated with survival, suggesting potential prognostic indications and a portable test for TBI [
41]. Similarly, immunohistochemical evidence of diffuse axonal injury (periventricular region only) and blood–brain barrier breach (both periventricular and cerebellar regions) were also present, with previous research suggesting a close coupling between these pathologies [
42‐
44]. In contrast, there were no significant differences between the Placebo and Sham cohorts on an immunohistochemical marker of inflammation (IBA1) following correction for multiple comparisons. The lack a neuroinflammatory response most likely reflects the relatively brief, 5-h post-injury monitoring period employed in the current study, as neuroinflammation has been shown to be present for multiple years post-injury following TBI [
45].
Estrogen sulfate has been shown to increase cerebral perfusion pressure, increase partial brain oxygen pressure and decrease intracranial pressure, but not to affect markers of diffuse axonal injury in a previous rodent study [
30]. Contrary to our a priori predictions, EE-3-SO
4 showed evidence of normalizing plasma levels of Aβ40 rather than biomarkers traditionally associated with blood brain barrier breach or neuroinflammation. Aβ is a 40–42 amino acid long peptide generated by successive cleavage of amyloid pre-cursor protein by β-secretase followed by γ-secretase [
44]. Although Aβ42 is believed to be more toxic, both forms have been shown to be rapidly released post-TBI, persist for weeks to months post-injury, and are typically viewed as potential markers of diffuse axonal injury [
46,
47]. Numerous preclinical studies have suggested neuroprotective effects for 17β-estradiol [
48], although estradiol is also elevated post-TBI and has been shown to confer an increased risk of death in severe human TBI [
49]. However, it remains unknown whether the elevated levels of estradiol post-TBI are due to decreased metabolism (i.e., hydroxylation of estradiol to estrone or increased synthesis due to increased aromatase activity). Although promising, current findings of a more rapid recovery in plasma Aβ40 following EE-3-SO
4 administration require further replication given the lack of efficacy for other female steroidal hormones in clinical TBI trials [
50] and current null findings for APP immunohistochemistry.
In the current study, male sex was associated with a nearly twofold increase in mortality rate regardless of drug assignment. There is a rich preclinical literature suggesting that biological sex and associated female endogenous steroidal hormones affect systematic responses to both blunt force and neurotrauma, but with mixed findings in clinical studies [
9,
40,
48,
51‐
53]. Specifically, retrospective clinical studies suggest that female sex may be protective against blunt-force trauma complications such as organ failure and sepsis rather than confer a benefit in terms of mortality [
53‐
55]. Other clinical studies have suggested that only perimenopausal or postmenopausal females demonstrate decreased mortality following isolated moderate to severe TBI [
56,
57], whereas pediatric-focused TBI studies indicated increased survival only for post-pubescent females [
58,
59]. The latter more closely corresponds to the approximate age of the swine used in the current study and potentially suggests a U shaped relationship between female sex and neuroprotection as a function of age.
There are several limitations to the study that should be noted. First, the current study purposefully did not measure several physiological functions (cerebral perfusion pressure, partial brain oxygen pressure, intracranial pressure, etc.) due to their invasive nature. The study design was intentionally focused on point-of-care and blood based biomarkers that can readily be performed in humans relative to more sophisticated immunohistochemical assays, and our aim to examine a more realistic closed head injury (i.e., intact skull). Blood samples and embedded brain tissue from this study will be made available upon request for additional, secondary analyses. Second, all animals received anesthesia throughout the entire protocol in compliance with the approved ethical framework for this study. Although this is unlikely to have influenced drug-related outcomes due to the fully randomized and blinded design, it may have artificially inflated mortality rates associated with respiratory depression across both cohorts. The selected anesthetic regimen partially mitigated this confounder through utilization of agents that minimize respiratory depression (i.e., midazolam and ketamine) relative to isoflurane, but in doing so also potentially increased neuroprotective effects [
60]. Finally, animals were only monitored for up 5 h post-injury in the current study, which limits the conclusions that can be drawn about more long-term therapeutic effects of EE-3-SO
4 or long-term pathophysiological consequences of the TBI + HS model.
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