Background

The serious consequences of traumatic brain injury (TBI) have been naively minimized or suppressed for years. Not long ago it was easy to cast TBI as nothing serious, when delivering a “knockout” is the ultimate goal for a boxer, or a “good hit” marks a talented defensive back in American football. Indeed, TBI was euphemistically described as “shell shock” in the First World War and was often a pejorative for the victim’s character. However, more recently awareness in two areas has dramatically changed public perceptions for the consequences of TBI.

First, the exposure of our troops to the asymmetric warfare in the Middle East, with widespread use of improvised explosive devices (IED) has spotlighted TBI. These simple, inexpensive, yet brutally effective weapons have dramatically increased warfighter’s TBI cases ( ). IED explosions have caused the greatest number of injuries in Iraq and Afghanistan as compared to other large-scale conflicts ( ). This weapon’s blast wave-initiated brain damage (ie, closed head) is the most common source of TBI induction, where pressures can approach 1000 atm, with damage at a microscopic or subcellular level ( ). The extent of blast injury has not only raised TBI awareness but has also sent many TBI patients home to the United States. Estimates are that between 2001 and 2007, 320,000 returning veterans from Iraq and Afghanistan were TBI positive ( ). Sadly, while brain-injured warfighter’s numbers have expanded enormously, there are no effective pharmacological treatments for TBI, despite the fact that a great deal of effort has been expended examining various therapeutic agents for TBI, including progesterone ( ). Putting it bluntly, stated: “To date, there is no specific drug treatment for acute brain injury, and many seemingly promising agents emerging from pre-clinical animal models have failed in clinical trials.”

The second area promoting TBI awareness is sports-related injuries, which focus our attention when children or celebrity athletes are involved. While military TBI is most likely to be induced by blast-wave forces ( ), civilian TBI is more often the result of blunt trauma and concussion ( ). Despite different inductions, there is likely to be a substantial overlap for military and civilian TBI ( ), where both can trigger downstream neurodegenerative disease, increasing with injury severity. However, severity may be a misleading criterion per se. While the military injuries from blast waves are often classified as mild TBI, this “mildness” blurs with repetition of the insults ( ). The same can be said with sports-related mild TBI, where repeated blows to the head often lead to serious consequences, such as chronic traumatic encephalopathy or Parkinson’s disease ( ), sometimes arising years later.

The Golden Hour

Effective therapeutic treatments for trauma must be administered as soon as possible postinjury. This has promoted the “golden hour” concept in trauma care, a window of opportunity to optimally deliver life-saving treatment. The golden hour rule has been clearly validated for TBI by the Resuscitation Outcomes Consortium ( ). It was found that:

Among out-of-hospital trauma patients meeting physiologic criteria for shock and traumatic brain injury, there was no association between time and outcome. However, the subgroup of shock patients requiring early critical resources and arriving after 60 minutes had higher mortality.

One means to provide early TBI treatment is to enable delivery by first responders. Thus the treatment should be highly portable, space efficient, stable, and convenient to administer.

Mechanisms of Estrogen Action

Estrogen has multiple modes of action, which can be influenced by route of delivery and solubility of the estrogen hormone. It is well known that estrogen acts via nuclear receptors, where estrogen ligands entering the cytoplasm engage estrogen receptors (ER), inducing dimerization and migration into the nucleus, whereupon ER activate a variety of genes via genomic estrogen response elements ( ). There are two ER isoforms, ERα and ERβ, which are expressed with varying levels and tissue specificities throughout the body, for both females and males. A third ER exists in the form of a G protein-coupled receptor, initially named GPR30, but now called GPER. GPER is bound to the endoplasmic reticulum and initiates rapid “nongenomic” actions ( ). GPER also has the ability to mitigate experimental TBI ( ).

ER also exists within a complex of signaling proteins (ie, signalosome) on the plasma membrane. When engaged, these receptors are major effectors of the rapid, nongenomic response ( ). We feel that this latter response is most likely the initial pattern for exogenously administered E2, since physiological benefits are seen almost immediately after introduction of E2 ( ). Through the use of single photon emission computed tomography-computerized tomography (SPECT-CT), we have shown that estrogen enhances cardiovascular performance by increasing cardiac output and kidney and liver perfusion in the face of severe hemorrhage ( ). Interestingly, the level of blood in the brain was not increased for E2 treatment over vehicle controls. Perhaps this is not such a paradox, since the rats are anesthetized to keep them still for imaging, reducing the brain’s demand for blood. In addition, trauma-hemorrhage (T-H) rats are in shock after 60% blood loss, which further reduces brain metabolism. It should be noted that in all our experiments, E2 is administered an hour or more after the injury, be it TBI or T-H.

The Neuroprotective Effects of Estrogens

There are several published accounts that document the neuroprotective properties of E2 ( ), which suggest potential for therapeutic use. Indeed, E2 is intrinsic to the brain, with local production in both males and females. This brain-endogenous E2 is neuroprotective as well ( ).

Engler-Chiurazzi and his colleagues have published reviews detailing the breadth of E2’s benefits for cognitive aging and brain injury ( ), including a historical review of two decades of research specifically for Alzheimer’s disease and stroke ( ). Of particular interest in this latter paper is findings from a dissection of the estrogen molecule to learn what is essential to confer neuroprotection, using a large library of estrogenic derivatives. Substitutions on the steroid A ring of alkyl groups at the 2 or 4 carbon positions exhibited enhanced neuroprotection from cerebral ischemia, which was brain-specific, acting without stimulating peripheral tissues ( ).

Estrogen replacement therapy’s risk/benefit profile in postmenopausal women remains controversial, especially in light of the higher incidence of stroke in these women ( ). These authors found that the hitherto unexplored effects of E2 on astrocytes suggests promise for neuroprotection based on a murine model, which mimics estrogen replacement therapy in ovariectomized mice. This effect was found to be mediated via ERβ of astrocytes ( ). There is also a crucial role for astrocytes in stroke ( ), where astrocytes respond to ischemia with reactive gliosis, excitotoxicity, and neuroinflammation.

The brain is unique among organs because, owing to the blood–brain barrier, it is essentially a “closed” system where estrogen can be synthesized and used locally. For example, it has been found that hippocampus-produced E2 maintains long-term potentiation and thus synapse health in females, but not males ( ).

A fascinating example of the neuroprotective actions of endogenous gonadal estrogens has been explored by Azcoitia et al. in a neurotoxicity model ( ). If the excitotoxin kainic acid is injected systemically at low doses, it can cause hippocampal hilar neurons to degenerate. However, injecting kainic acid in female animals at the beginning of estrus (ie, maximum systemic estrogen level) produced no neuronal damage, while injection at proestrus (lowest estrogen level) caused loss of hilar neurons, as did injection into ovariectomized females. Similarly, intact males suffered no damage from kainic acid, while castrated males lost hippocampal neurons. This seeming paradox may reflect the conversion of testosterone into estrogen by aromatase in vivo. The importance of early delivery was noted. When E2 was injected concurrently with kainic acid into ovariectomized females, neuronal damage was prevented, but with a delay of 24 h, there was loss of hippocampal neurons ( ). Again exploiting a kainic acid toxicity model, it was found that selective estrogen receptor modulators (SERM) exhibit some similar benefits, but overall E2 was superior. For example, E2 substantially downregulated active gliosis, which was less efficacious with SERMs ( ).

Neuronal Stretch Assay

The neuronal stretch assay is an excellent in vitro correlate for TBI mechanics, as the injured brain experiences shear, stretch, and compression. Furthermore, the assay reduces use of rats by deriving multiple assays from a small number of rat pups. This assay relies on the growth of explanted newborn rat neurons in tissue culture, grown on a flexible silastic membrane. This apparatus precisely and reproducibly deforms the membrane momentarily, producing the “stretch” injury.

Multiple Therapies from One Drug

In light of the foregoing, three significant points are noteworthy. First, we can extend life in animals from otherwise universally fatal 60% hemorrhage to 6 h with E2 and no other intervention, including resuscitation, suggesting that early E2 administration may extend the golden hour. This could be of great value to treat TBI from the battlefield or remote civilian areas. Second, if E2 is administered as a therapeutic drug for obvious severe hemorrhage as part of a polytrauma cluster, it should likewise be treating the pathologies of TBI in a “piggyback” fashion, where TBI may occur concurrently with blood loss and other injuries. Third, while we will not discuss sepsis here, we nonetheless have a basis to suggest that administration of E2 may mitigate the threat of sepsis in injured patients. This has been demonstrated in rat models which mimic penetrating wounds with necrosis, where the cecum is perforated to release gut flora into the peritoneal cavity, and ligated to induce necrosis ( ). In our studies, E2 administration after hemorrhage was followed by sepsis induction by cecal ligation and puncture. E2 produced improved immune function, cardiac output, splanchnic perfusion, and oxygen utilization ( ). A diagram of the possible “triple therapy” is seen in Fig. 10.7 .

Formulating a Drug

Our experimental treatment relies on a soluble form of 17β-estradiol, enabling administration of the drug i.v. or intraosseously. We have examined microencapsulation with β-cyclodextrin, as well as mono- and disulfate-conjugated E2. While all were efficacious, none were available in a good manufacturing practice (GMP) form, as required by the FDA. We were also advised to explore the synthetic estrogen 17-α ethinyl estradiol (EE) with a sulfate at the 3 carbon position (EE-3-SO ₄ ). EE has been in use for decades, being originally synthesized in 1938, and subsequently used to produce more potent contraceptives ( ), as well as being applied to hormone replacement therapy ( ), with an excellent safety record for both applications. Thus by using this synthetic form of estradiol, we could increase the drug’s efficacy. We have been granted a patent for the EE-3-SO ₄ drug and are pursuing an FDA application for an investigational new drug (IND). Preclinical experimentation is under way for both TBI and severe hemorrhage in our laboratory and with collaborators.

Estrogen and Traumatic Brain Injury