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Laskowitz D, Grant G, editors. Translational Research in Traumatic Brain Injury. Boca Raton (FL): CRC Press/Taylor and Francis Group; 2016.

Chapter 11Translational Principles of Neuroprotective and Neurorestorative Therapy Testing in Animal Models of Traumatic Brain Injury

Authors

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INTRODUCTION

It is estimated that there are in excess of 1.5 million cases of traumatic brain injury (TBI) each year in the United States. Of these, 1.2 million seek medical care. Out of this number, approximately 90% of TBIs are “mild” in severity (Glasgow Coma Score = 13–15) although some require brief hospitalization, and the neurological and psychological consequences are often life changing. The other 10% of TBIs result in either severe (Glasgow Coma Score = 3–8) or moderate (Glasgow Coma Score = 9–12) acute sequelae and typically require intensive medical treatment and extended recovery periods. Although TBI can victimize active individuals at any age, most occur in young adults in the second and third decades of life. Moreover, the majority of TBI patients now survive their neurological insults due to improvements in emergency, neurological intensive care, and surgical treatments. Nevertheless, the need for intensive rehabilitation and the reality of prolonged disability exacts a significant toll on the individual, his or her family, and society. Effective therapies to either attenuate post-TBI secondary damage to brain tissue, thereby preserving the anatomic substrate needed for recovery of neurological function, or that enhance the functional capacity of the surviving brain tissue, represents one of the most pressing unmet needs in medicine to improve the outlook for those with TBI by enabling higher levels of independence and productivity and a reduction of the costs of longterm care to the injured individual, their families, and society.

The goal of this chapter is to discuss the importance of preclinical evaluation of potential therapies for TBI in animal models that mimic the human disorder as a prelude to the translation of these into clinical trials. The focus will be on a brief description of the various rodent (mouse and rat) TBI models, what subtypes of TBI they mainly model, and the presentation of basic principles of preclinical therapeutic testing. For a more detailed discussion of the specifics of particular TBI models, the reader is referred to the several excellent in-depth reviews on TBI modeling that have been published during the last decade.1–8 The discussion of how to test therapies in TBI, or TBI-relevant models, will be conveyed primarily in relation to pharmacological therapies. However, many, if not all of the principles that define a thorough preclinical evaluation of drugs in animal models are in fact equally applicable to the application of therapeutic hypothermia as well as gene and cellular transplant therapies.

GOALS OF DRUG THERAPIES FOR ACUTE TRAUMATIC BRAIN INJURY (TBI)

Neuroprotection

Much of the opportunity for pharmacological intervention to preserve neurological function after acute TBI is based on the fact that most of the vascular and/or neurodegeneration that follows these injuries is not due to the primary mechanical (i.e., shearing of blood vessels, and nerve cells) insults, but rather to secondary molecular and pathophysiological injury events set in motion by the primary injury. Indeed, during the first minutes, hours, and days following injury, a neurodegenerative process is initiated that is proportional to the magnitude of the initial insult. Nevertheless, the initial anatomical continuity of much of the injured brain tissue in the majority of cases, together with our present knowledge of many of the factors involved in the secondary injury process, has lead to the notion that pharmacological treatments that interrupt the secondary cascade, if applied early, could improve brain tissue survival (preservation of axons, nerve cell bodies and synaptic connections), and thus preserve the necessary anatomic substrates for functional recovery to take place. The pharmacological strategy of interfering with acute (first 72 hours) and subacute (72 hours to 14 days) post-TBI neuronal, axonal, and synaptic loss is referred to as neuroprotection.

In particular, a key determinant in neurological recovery after TBI concerns the secondary loss of axons and consequently the lost connection between neuronal cell bodies, the distal nerve terminals that make synaptic connections with downstream neurons. This often widespread loss of axons in the injured brain is referred to as diffuse axonal injury (DAI) or traumatic axonal injury (TAI), much of which is due to a potentially interruptible process involving localized disruptions of axonal ionic balance; intra-axonal accumulation of calcium (Ca++), which is exacerbated by free radical-induced oxidative damage to Ca++ homeostatic mechanisms; mitochondrial failure, and the triggering of Ca++-activated calpain-mediated proteolysis of axonal neurofilaments.9–14 Accordingly, various compounds that attenuate these secondary injury processes have been documented to reduce axonal loss. However, it should be realized that a significant factor in influencing the extent of neural injury is a decrease in brain microvascular perfusion (i.e., secondary ischemia). When this occurs, the result is an exacerbation of the injury process due to superimposed brain tissue ischemic hypoxia. Moreover, deficiencies in brain cerebral blood flow (CBF) can be aggravated by systemic hypotension and hypoxia, which are well documented causes of outcome worsening after TBI. Thus, it is important to note that secondary injury involves both parenchymal and microvascular events, and that some neuroprotective compounds can work by protecting brain parenchymal neurons and glia directly or indirectly by improving cerebral perfusion pressure (CPP), CBF, and microvascular oxygen delivery that is required for maintaining cellular respiration and tissue viability.

Neurorestoration

The second approach to the treatment of TBI involves attempting to restore lost neurological function once the extent of the traumatic or ischemic damage to the brain and the associated neurological deficits has stabilized. Until a decade ago, it was firmly believed that once the brain (or spinal cord) was damaged by the secondary injury process, there was little, if any, capability for regeneration of axons and formation of new synapses to take the place of the lost neural elements. However, over the last several years, it has been discovered that the brain is indeed capable of significant structural and functional repair, plasticity and regeneration that can be pharmacologically enhanced. Approaches for accomplishing this include reawakening the growth potential of the surviving neurons or antagonizing the multiple inhibitory factors that have been discovered whose activity is aimed at inhibiting axonal growth and synaptogenesis. Alternatively, cellular replacement may be achievable in certain brain regions that possess nascent neural stem cells. It is increasingly apparent that these endogenous stem cell populations in the injured brain might be pharmacologically stimulated to divide, migrate, and differentiate into neuronal or glial precursor cell types, and ultimately neurons and remyelinating oligodendroglia, respectively. To enable this process, the molecular mechanisms that control neurogenesis and gliogenesis are rapidly being revealed providing targets for pharmacological neurorestoration of function. Several pharmacological mechanisms have been identified that can be targeted to try to enhance the function and/or structural plasticity of neuronal pathways that survive the ravages of post-traumatic secondary injury.15–18

PREVIOUS TBI CLINICAL TRIALS OF NEUROPROTECTIVE AGENTS AND WHAT WE LEARNED RELEVANT TO THE NEEDS FOR PRECLINICAL DRUG EVALUATION IN ANIMAL MODELS

In the early 1980s, several pharmaceutical companies became attracted to the idea of discovering neuroprotective drugs for the acute treatment of TBI and stroke. As a result, several compounds were discovered that were entered into development with some making their way into large, double-blind, multicenter phase III clinical trials in TBI. These efforts, which actually began in the late 1970s and continued into the early 2000s, were primarily directed at three general pharmacological mechanistic strategies to interrupt secondary injury processes: (1) reduction of intracellular calcium overload (L-type calcium channel blockers), (2) inhibition of glutamatemediated excitotoxicity (i.e., glutamate receptor antagonists), and (3) interruption of reactive oxygen-mediated damage (i.e., free radical scavengers/antioxidants).

Calcium Channel Blocker Nimodipine

Accumulation of intracellular calcium is a major player in secondary injury after TBI. One of the mechanisms for the postinsult calcium overload involves depolarization-induced entry via voltage-dependent L-type channels. Accordingly, the first neuroprotective approach to be tested in Phase III clinical trials in TBI was the competitive L-type calcium channel blocker nimodipine, which was entered into clinical trials beginning in the late 1970s. In two different Phase III multicenter TBI (moderate and severe) trials,19 no overall benefit was revealed. However, retrospective analysis of the TBI trials revealed that nimodipine may have improved outcome in patients with traumatic subarachnoid hemorrhage (tSAH).19 This is not an insignificant finding since about half of all patients with severe TBI have tSAH as part of their post-traumatic pathology, which carries with it a largely vascular pathophysiology including severe blood-brain barrier (BBB) compromise, loss of microvascular autoregulation and spasm of major cerebral arteries that restricts CPP and CBF. Furthermore, nimodipine has been shown to produce a slight but significant increase in survival in aneurysmal SAH patients20 and was approved by the U.S. Food and Drug Administration, and in most countries for the treatment of this condition. While its neuroprotective efficacy is quite limited, nimodipine represents the first agent to be approved for neuroprotective use even though much of its effect is probably mediated via protection of the microvasculature and vasodilation-mediated improvements in CBF. However, due to its dose-related vasodilatory action, the compound must be used with care since it can lower arterial and cerebral perfusion pressures, which can potentially exacerbate post-TBI secondary brain injury. In any event, we now know that prolonged opening of the voltage-dependent L-type calcium channel is only one of the mechanisms for post-traumatic intracellular calcium overload, and probably much less important than the others including glutamate receptor overstimulation-induced “excitotoxicity” and oxidative damage-impairment of the multiple intracellular mechanisms responsible for maintaining calcium homeostasis.21

Glutamate Receptor Antagonists

In order to inhibit post-TBI glutamate-mediated excitotoxicity, multiple glutamate receptor antagonists were taken into Phase II and III trials including the competitive N-methyl-D-aspartate (NMDA) receptor antagonists selfotel (CGS 19755) and aptiganel (CNS 1102) that block the binding of glutamate to its receptor complex recognition site, eliprodil that blocks the polyamine site, and CP-101,606 (traxo-prodil) that blocks the NR2B subunit on the NMDA receptor complex.22 None of these produced a statistically significant improvement in neurological recovery in TBI patients, although traxoprodil came close (p < 0.1) to producing a significant improvement in favorable outcome in a relatively large (400 severe TBI patients) phase II clinical trial.23 In retrospect, there was precious little literature generated concerning the testing of the competitive NMDA antagonists in TBI animal models before the aforementioned glutamate receptor antagonists were placed into clinical development. Much of the rationale for trying them in clinical TBI was based upon their efficacy in ischemic stroke models and the common notion that whatever was neuroprotective in a stroke model would also be effective in TBI paradigms. More important, whether they were tested in stroke or TBI models or both, the published work involved very early postinjury drug administration times of usually less than one hour. There was little systematic definition of the therapeutic window (i.e., how long after TBI could the treatment be delayed and still produce neuroprotective efficacy), but what was done showed that neuroprotective efficacy was lost during the first hour after injury. Nevertheless, the apparently short therapeutic window for NMDA antagonists in animal models did not dissuade companies from conducting trials of NMDA receptor blockers allowing treatment initiation as much as 8 hours after TBI. This was due to the prevailing assumption among TBI (and stroke) researchers and clinicians, that whatever the therapeutic window might be in rodent animal models, it would probably be considerably longer in humans. In other words, since mice and rats are hypermetabolic compared to humans, the progression of post-TBI secondary injury and neurodegeneration would be more rapid and would need to be treated with a neuroprotective agent quicker than in humans. However, the only evidence that supports this assumption is from microdialysis studies of glutamate release in injured rat brain showing that the massive injured-triggered release of glutamate only last for minutes, whereas in human TBI patients, the duration of elevated glutamate release may go on for 6 hours to several days.24 In contrast to the older microdialysis studies of glutamate-release kinetics in TBI models, more recent results in the rat central fluid percussion model using real-time continuous microelectrode monitoring of extracellular glutamate shows that the injury-induced increase in glutamate levels in injured rat cerebral cortex in fact persists for days,25,26 as it does in human TBI. Therefore, if the duration of glutamate release in both rodents and humans is prolonged for days, then why would rodent TBI and stroke models only show a NMDA receptor antagonist neuroprotective therapeutic window of less than an hour?

Following the failures of the NMDA antagonist clinical trials conducted in TBI patients, Shohami and colleagues27 revealed in their mouse weight-drop TBI model that the short therapeutic window for these compounds may be due to the fact that the initially glutamate overstimulated NMDA receptors in the injured brain actually enter into a hypofunctional state by an hour after TBI, and that this loss of function persists for days. Consequently, blocking the receptors after the first hour with the NMDA receptor blocker is no longer useful since the NMDA receptor is already in a nonfunctional state.27 These investigators additionally showed that what was needed in regard to NMDA receptor modulation was to administer NMDA to reactivate the NMDA receptors beginning at 24 or 48 hours after TBI and that this lead to an improvement in 14-day cognitive performance. Subsequent experiments have shown that this can be more safely achieved by administering a weaker NMDA receptor agonist or a partial agonist such as D-cycloserine,28 which is actually in early clinical trials in TBI patients as a neurorestorative approach to improve long-term post-TBI recovery. In summary, while few, if any, investigators would question the important role of glutamate NMDA receptor activation in the initiation of excitotoxic brain damage after TBI, the efficacy of NMDA receptor blocking as a neuroprotective strategy is most likely clinically impractical due to a short therapeutic window, whereas what may be more useful is to pharmacologically reactivate brain NMDA receptors in the postacute recovery phase after TBI.

Free Radical Scavengers/Antioxidants

In the case of efforts to interrupt reactive oxygen damage, the polyethylene conjugated form of the superoxide radical scavenger Cu/Zn superoxide dismutase (PEG-SOD) was evaluated in trials conducted in moderate and severe TBI patients. Although it showed a positive trend in an initial, small phase II trial,29 subsequent phase III trials failed to show any enhancement of neurological recovery.22

A much bigger development program was undertaken with the 21-aminosteroid lipid peroxidation inhibitor tirilazad. Tirilazad was extensively evaluated in animal models of TBI as well as SCI, ischemic stroke, and subarachnoid hemorrhage, and shown to exert a variety of neuro- and vasoprotective effects.30,31 Based upon these preclinical studies, clinical trials of tirilazad were conducted in TBI,22,32 SAH,33 ischemic stroke34,35 and SCI.36 In TBI, an initial North American trial of 1100 patients, comparing tirilazad treatment with a placebo for 5 days initiated with 4 hours postinjury, ended with such a confounding randomization imbalance that no meaningful efficacy analysis could be extracted. In contrast, a European phase III trial was successfully completed, but it failed to show an overall effect in moderate and severely injured patients. However, post-hoc analysis revealed that the compound significantly improved survival in both moderately and severely injured male patients with traumatic SAH.32 This beneficial effect in the tSAH subgroup, which represents about half of severe TBIs, was not surprising in that the drug had previously been shown to improve recovery and survival in a Phase III trial in aneurysmal SAH patients.33 Interestingly, this effect in tSAH and aneurysmal SAH was mainly apparent in male patients. This gender difference was found to be partially due to a faster rate of tirilazad metabolism in females. Nevertheless, subsequent female-only trials with higher tirilazad doses that were calculated to duplicate the exposure levels in males did not reveal the same level of efficacy as seen in male patients, although beneficial effects were apparent in the more severe SAH females.37,38 Thus, the issue of gender differences in neuroprotective drug responsiveness clouds the interpretation of tirilazad’s neuroprotective efficacy.

Learnings

This brief history of neuroprotective drug discovery and development over the past 20–25 years could be fairly characterized as a series of often high profile and expensive failures. Although these have largely dampened the enthusiasm of the pharmaceutical industry for this therapeutic area, much has been learned from them that could, and should, serve as a roadmap for future efforts aimed at pharmacological neuroprotection and improved neurological recovery after TBI. Postmortem analyses of mistakes made in TBI drug development have been published, and a careful reading of them reveals a host of shortcomings in past preclinical testing of candidate neuroprotective agents and in clinical trial design and conduct that need to be addressed in the future.22 A summary is provided in Table 11.1.

First, the discovery of the first generation of neuroprotective agents, which included glutamate receptor antagonists, calcium channel blockers, and free radical scavengers/antioxidants, occurred prior to the elucidation of an adequate understanding of the intricacies of the targeted secondary injury mechanisms. In each case, there was inadequate knowledge of the time course and interrelationships of these events; their therapeutic windows for effective treatment intervention; and how these were either similar or different between species, injury models, genders, and between animals and humans. In the case of reactive oxygen mechanisms, our knowledge of the key ROS species and their sources or cellular origins and targets was insufficient to guide the design of optimum antioxidant neuroprotective compounds. Second, the preclinical efficacy testing of compounds was often naively deficient and failed to consider the pathological complexity of human TBI and the need to test potential neuroprotective agents in multiple models that might define whether a particular agent might be broadly used across the full TBI spectrum or for a specific TBI phenotype as discussed in the following section. Third, a major problem was that the clinical trials were poorly designed in regard to a failure to allow the preclinical TBI animal models results (e.g., therapeutic window) to inform the clinical trial design, inclusion of all types of TBI pathological phenotypes to be in the trial, and lack of standardization of acute treatment guidelines and neurorehabilitation protocols across the patient enrollment sites.

VARIABILITY IN TBI PATHOLOGY AND PATHOPHYSIOLOGY: RATIONALE FOR TESTING NEUROPROTECTIVE OR NEURORESTORATIVE DRUGS IN MULTIPLE TBI MODELS

As eluded to at the beginning of this chapter, the traditional clinical assessment tool for expressing the severity of TBI in patients is the Glasgow Coma Scale (GCS) score, so named because of its development in the mid-1970s by two prominent TBI-focused neurosurgeons in Glasgow, Scotland, Brian Jennett and Graham Teasdale.39,40 The scale involves scoring of three responses to either a painful stimulus or to command: eye opening, verbal response, and basic motor response. Eye opening is worth a maximum of 4 points: 1 for no opening in response to pain or to command, 2 for eye opening in response to pain, 3 for eye opening in response to a verbal command, and 4 points for spontaneous eye opening. Verbal response is worth a maximum of 5 points: 1 for no response, 2 for a response to sounds, 3 for a nonsense response, 4 for a confused response, and 5 for the ability to carry on a conversation with the examiner. The basic motor response in response to a pain stimulus is worth a maximum of 6 points: 1 for no response, 2 for a generalizing response to the pain, 3 for arm extension, 4 for arm flexion, 5 for a localizing response to the pain (ability to identify where the pain is emanating from), and 6 if a motor response occurs on command. The total GCS ranges from a low score of 3, which would be a person who has suffered a TBI and is in a deep coma with no eye opening, verbal, or motor response, to a full score of 15, which is a patient who is has full normal responses. The scoring range is typically divided into three severity categories: 3–8 for someone who is said to have suffered a “severe” TBI, 9–12 for a “moderate” TBI, and 13–15 for a “mild” TBI. Someone who is at 8 or below is characterized as being comatose in contrast to someone who has improved to a 9 who is considered to be emerging from a coma. It should also be remembered that while scores will increase as the TBI patient improves his or her neurological status, it is also not uncommon for the GCS to decrease from an initially moderate or even mild level down to the severe level. Clinicians who treat acute TBIs refer to patients who are initially responsive when first admitted to the emergency department, but subsequently slips progressively into a coma as falling into a category of TBI patients who “talk and deteriorate.” Such patients demonstrate the influence of progressive post-traumatic secondary pathophysiological changes (e.g., brain swelling, decreased cerebral blood flow, hemorrhage expansion) that are taking place during the first minutes or hours after TBI.

To illustrate the variability in TBI-induced pathology, Figure 11.141 displays the computerized tomography (CT) scans of six acutely severe TBI patients who all have a GCS <8. Yet, each patient reflects differing post-traumatic pathological manifestations caused by their TBIs. The top left CT shows an epidural hematoma (EDH), which by itself can lead to a good outcome if it is promptly surgically evacuated and additional hemorrhage stopped. However, because they are generally caused by traumatic meningeal arterial hemorrhage as a result they can expand rapidly raising intracranial pressure (ICP) and patient death due to tentorial herniation and resulting brainstem compression. A patient who sustains a TBI and presents in the emergency room able to talk, but with a rapidly developing EDH is perhaps the most likely to talk and deteriorate. The bottom left CT scan shows a subdural hematoma (SDH), which is from ruptured cerebral veins that may more slowly increase in their dimension over time. If small, they may be missed on the first CT and thus not surgically removed during the acute post-TBI period. However, if it grows slowly it will begin to increase ICP and cause neurological deterioration. In both of these patients, the CT reveals the hematoma being large enough to press the midline structures toward the opposite side of the brain.

The middle top CT in Figure 11.1 is from a TBI patient who has manifested a focal brain contusion as well as an SDH on the left side of the CT. The middle bottom CT is from a TBI patient with a traumatically induced subarachnoid hemorrhage (SAH) along with an SDH on the left side of the CT and a smaller intraventricular hematoma (IVH) on the right side of the CT. TBI patients with hemorrhagic contusions, SAHs, and IVHs, if they are large, typically have the worst prognosis because these pathologies have a good chance of producing blood-brain barrier compromise and rapidly evolving vasogenic brain edema, and acute and chronic arterial vasospasm compromising cerebral blood flow (CBF) and brain oxygen delivery. On the bottom right of Figure 11.1 is a CT from a patient with no apparent hemorrhagic manifestations but has massive diffuse swelling as judged from the inability to discern visible cortical gyri and sulci or ventricles since the swelling has closed them off. Finally, in the top right CT is a TBI patient in which the cortical structure is largely intact, the midline structures are not shifted, and no hemorrhagic pathologies are visible. This type of patient who was verified to have a TBI and is displaying neurological sequelae as a result is referred to as having diffuse axonal injury (DAI). However, each of the patients in Figure 11.1 will have DAI along with their other pathologies.

To further illustrate the variability in TBI pathologies and pathophysiologies, Tables 11.2 and 11.3 list the post-traumatic pathologies seen in a recent series of 17 patients (13 males, 4 females) who presented at the University of Kentucky Medical Center with initial post-TBI GCS scores between 4 and 10. The most frequent pathology seen in 7/17 patients (41.2%) was traumatic SAH. However, what is apparent from the tables is that the typical severe or moderate TBI patient can have multiple post-traumatic pathologies. In other words, the pathology and associated pathophysiology of individual TBIs is highly variable even though their admission GCS values are all in the same range.

Similarly, a prominent group of TBI experts from the Departments of Neuropathology and Neurosurgery associated with the University of Glasgow Institute of Neurological Sciences published in 2011 a summary of neuropathological findings in a group of 85 TBI victims treated at their center who survived at least a month after their TBIs but subsequently died. As assessed by the Glasgow Outcome Scale (GOS),42 the 85 at the time of their deaths were either in a vegetative state (35), severely disabled (30), or moderately disabled (20). Neuropathological assessment of their brains revealed that 84% had cerebral contusions, 58% had DAI, 67% had ischemic brain damage, 68% had bilateral enlargement of the ventricles indicative of widespread brain atrophy reflecting extensive neurodegeneration, 52% had thalamic damage, and 55% showed evidence of raised ICP. Moreover, 41% had undergone surgical evacuation of an intracranial hematoma (either EDH, SDH, IVH, IPH, or SAH). Of these variable pathologies, DAI, raised ICP, thalamic damage, ventricular enlargement, and ischemic brain damage were associated with the worse outcomes. In particular, diffuse or multifocal primary axonal injury or secondary ischemic damage was associated with the most severely impaired outcomes after a TBI. These statistics from Adams et al.,42 as well as those in Tables 11.2 and 11.3 from the University of Kentucky, underscore the complexity and variability of post-TBI secondary injury-associated neuropathologies. Accordingly, this reality has necessitated the development of multiple preclinical TBI paradigms in order to model the different TBI phenotypes and more importantly for exploring the efficacy of neuroprotective and neurorestorative treatments for each injury type.

TRAUMATIC BRAIN INJURY AND TRAUMATIC BRAIN INJURY-RELEVANT MODELS

As pointed out earlier in this chapter, several excellent and usually comprehensive reviews on animal TBI models have been published in the past 15 years.1–8,43 However, one the most recent reviews that is aimed at not only classifying the types of models, but discussing their pathological and pathophysiological characteristics as those that relate to particular TBI phenotypes and their appropriateness for preclinical neuroprotective and neurorestorative drug testing is adopted by the author for the present discussion.4 However, differing from the prior TBI animal model reviews, the current presentation additionally includes TBI-relevant models that while not involving an actual mechanical brain trauma are neurological injury paradigms that have relevance to particular TBI pathologies. These include SAHs, epidural hematomas, subdural hematomas, and ischemic focal stroke models that replicate some of the major sequelae of human TBI. The TBI and TBI relevant models are listed in Table 11.4.

Traumatic Brain Injury Models

In vivo TBI models include six basic types: diffuse, focal, mixed diffuse/focal, complex (e.g., diffuse or focal TBI complicated by either a hypotensive, hypoxic, or peripheral traumatic insult such as a tibial fracture), penetrating, and blast.

Diffuse TBI Models

There are four general types of diffuse TBI models. The first of these is the rat fluid percussion TBI paradigm that involves the application of a transient hydraulic pressure pulse onto the exposed dura mater over the midline of the brain, which is referred to as the central fluid percussion model. This model was first developed in the cat44 as well as pigs, dogs, and sheep, but later modified for use in rats45,46 and mice.4 It produces a widespread (i.e., “diffuse”) pattern of cellular and axonal neurodegeneration as recently shown using the de Olmos silver-staining method.47 The second is the rat impact-acceleration injury model developed by Anthony Marmarou at the Medical College of Virginia and hence often referred to as the Marmarou model. It involves a 0.5 or 1.0 kg weight-drop impact onto a helmet cemented onto the exposed skull. The acceleration component of the model is achieved by the sudden downward movement of the impacted head into a foam pad.48,49 The third is the mouse weight-drop concussion paradigm first developed by the current author50 and used extensively for neuroprotective drug testing.51–55 It involves dropping a 50 or 100 g weight at variable distance onto the exposed head of the hand-restrained mouse, which produces a diffuse brain injury along with either contracoup or lateral contusions in approximately a third of the mice.54 Although not widely used, it has been adopted by other laboratories for neuroprotective drug screening.55–58 More recently, other laboratories have developed mouse and rat weight-drop diffuse TBI models employing single or repetitive weight drop or pneumatic piston-induced injuries to the closed skull.1,59–63 The final category of diffuse TBI model used in the pig, cat, rabbit, or sheep, or primates involves induction of a rapid rotational acceleration of the constrained head that is particularly useful for induction of diffuse axonal injury.64

Focal TBI Models

For the induction of focal TBIs, there is the widely employed controlled cortical impact (CCI) model first developed by Lighthall and colleagues for use in ferrets to produce a contusion TBI65 that has since been adapted for use in rats66 and mice.67,68 It involves the infliction of a contusion injury through a small craniotomy via a pneumatically driven piston. The magnitude of the injury is generally varied by the depth of the cortical indentation (usually 0.5–1.0 mm in mice; 1.0–2.2 mm in rats). The CCI paradigm is a model of TBI-induced brain contusions. However, recent studies from the current author’s laboratory using the de Olmos silver-staining technique have demonstrated that the subsequent neurodegeneration is not as focal as generally thought.69,70 Widespread axonal degeneration is seen in not only the cortex, but also in the hippocampus and thalamus. Moreover, anterograde and retrograde axonal degeneration is also seen in the contralateral hemisphere, previously thought to be uninjured and thus incorrectly considered to serve as a contralateral control for the opposite injured hemisphere. Open skull weight-drop focal injury models have also been utilized in rats and mice.4 Last, a bilateral focal contusion model has also been used by Stein and colleagues.71

Mixed Diffuse/Focal TBI Models

The widely employed lateral fluid percussion TBI model developed in the late 1980s for use in rats72 represents a model that combines a cortical contusion in the deeper layers of the cerebral cortex underlying the fluid percussion site as well as more diffuse axonal degeneration in the ipsilateral as well as the contralateral hemisphere. Thus, it models the TBI mixed TBI phenotype seen in patients who have a cortical contusion as well as diffuse axonal injury. Together with the CCI model, the lateral fluid percussion TBI model is among the most widely used in experimental TBI research.

Complex TBI Models

The complex TBI models simply involve the use of one of the aforementioned TBI paradigms and exacerbating the pathophysiology of the mechanical TBI, whether it be a diffuse or focal type, by combining the injury with an episode of hypoxia or hemorrhagic hypotension. This is based upon the firmly established finding that if the acute TBI patient sustains a period of reduced oxygen delivery to the injured brain by suppressed respiration or by a reduction of blood volume due to traumatic hemorrhage or damage to the brainstem, cardiovascular, or respiratory control centers, that their outcome after their TBI will likely be worsened. Thus, combining the Marmarou impact acceleration, lateral fluid percussion, or the controlled cortical impact models with a defined episode of experimentally induced hypoxia (e.g., PaO2 <60 mm Hg) or hemorrhagic hypotension (e.g., systolic blood pressure <90 mm Hg or CPP <60 mm Hg) will predictably worsen the brain neurochemical and histological damage and exacerbate motor and cognitive recovery. Accordingly, this complexity provides a real-world relevant model of TBI complicated by a systemic insult that provides a larger hurdle for testing the efficacy of neuroprotective compounds. Similarly, a handful of investigators have begun to examine the combination of a polytraumatic insult involving a TBI complicated by a tibial fracture.73

Penetrating Ballistic-Like TBI Model

In the early days of TBI research, a few laboratories took on the task of developing models of penetrating TBI generally involving shooting a bullet into the brain of an anesthetized animal. However, shooting animals in the head was one of the lightning rods for attacks by animal welfare groups. Moreover, such models were difficult to control. However, since ballistic-induced penetrating TBI sustained by warfighters or by civilians who sustain criminal attacks, the need to model penetrating ballistic-like injuries is a significant need. Accordingly, Tortella and coworkers at the Walter Reed Army Institute of Research have recently developed and extensively characterized a highly controlled, nonbullet, penetrating ballistic-like brain injury (PBBI) model in rats.74–79 Furthermore, the same group has successfully tested a neuroprotective antiinflammatory compound, NNZ-2566, that is currently in early human development.80

Blast-Induced TBI Models

Over the past 13 years, since the beginning of the War on Terror, blast-induced TBI, it has become a growing focus in contemporary TBI research. Most of the blast models published to date involve the use of various “blast tube” devices to direct either the sudden triggering of a small amount of explosive or the sudden release of compressed air at the closed end of the blast tube to inflict a blast-induced TBI in a restrained rat placed near the opening at the opposite end of the tube. The pathology and pathophysiology of blast TBI is being widely studied by a number of laboratories as discussed in several recent papers and reviews.81–87

Traumatic Brain Injury Relevant Models

The chapter also includes a limited presentation of hemorrhagic and ischemic stroke models, because human TBIs, mainly on the more severe end of the spectrum, often include brain hemorrhagic or ischemic-hypoxic insults. This is due to post-traumatic vasogenic and cellular edema that causes a rise in intracranial pressure (ICP), which in turn decreases cerebral perfusion pressure (CPP) as explained by the simple equation: CPP = Mean arterial blood pressure (MAP) – ICP. Additionally, a decrease in MAP (i.e., systemic hypotension) due to hemorrhagic shock will also lead to a decrease in CPP. Whether due to increased ICP or decreased MAP, the fall in CPP will cause a secondary fall in cerebral blood flow (CBF) and oxygen delivery, which, if severe, can lead to an ischemic insult to the brain tissue on top of the mechanical TBI.

Intracerebral (aka Intraparenchymal) Hemorrhage Models

In regard to hemorrhagic stroke paradigms that are also relevant to the pathophysiology and neuroprotective therapeutic investigation of TBI, there are two types of insults to consider. The first is intracerebral hemorrhage (ICH), where cerebral blood vessels may be ruptured by the mechanical trauma with blood being released into brain parenchyma where it produces secondary brain damage by triggering brain edema (swelling) and mass effects that result in secondary ischemia within the tissue surrounding the intracerebral mass of blood (hematoma). For the induction of ICH, usually in rats, the approach is simply to inject a volume of the animals’ own blood directly into the brain parenchyma followed by an analysis of the volume of damage to the surrounding brain tissue.

Subarachnoid Hemorrhage Models

The second type of TBI-relevant hemorrhagic stroke model involves the induction of subarachnoid hemorrhage (SAH), where blood is released from mechanically ruptured vessels running through the subarachnoid space (between the subarachnoid and pial membranes covering the brain) from a burst congenital berry aneurysm ballooning out from one of the major arteries at the base of the brain. The pathophysiology of this type of hemorrhagic stroke also involves the triggering of a secondary ischemic insult due to the induction of delayed cerebral vasospasm that peaks in incidence at 4–7 days post-SAH. While both types of hemorrhagic insults are common in TBI patients, traumatic SAH (tSAH) occurs in approximately 50% of severe TBIs. To induce SAH experimentally, most of the available models involve the injection of a volume of the autologous blood, withdrawn immediately prior to SAH induction from the systemic circulation of the animal (e.g., rat, cat, rabbit, dog), into the subarachnoid space via injection into the cisterna magna or over one of the cerebral hemispheres via a small burr hole and puncture of the dura mater covering the brain. The most common endpoints for drug evaluation involve measurement of blood-brain barrier compromise, or decreases in cerebral blood flow during the first several post-SAH hours, or the assessment of cerebral vasospasm by histological or arteriographic methods between 2 to 7 days. The most sophisticated SAH model available involves the neurosurgical placement of an autologous blood clot around the base of the MCA in monkeys followed by arteriographic and histological ischemic damage measurements at 7 days.

Subdural and Epidural Hematoma Models

The rat lacks an arachnoid membrane, and thus the rat version of the SAH model involving injection of blood through the dura mater can also be thought of, and employed, as a subdural hematoma model. When the autologous, nonheparinized blood is injected through the dorsal burr hole through the dura mater, it will typically form a clot over the dorsal surface of the brain mimicking a post-traumatic subdural hematoma. Similarly, if the autologous blood is injected outside the dura, but beneath the skull it constitutes an epidural hematoma. Just as is done in the management of human post-TBI subdural and epidural hematomas, the experimental protocol involves surgical removal of the clot at a specified time followed by histological measurement of blood-brain barrier compromise and ischemic damage caused by the compression of the underlying brain tissue by the hematoma as well as the reperfusion injury caused by its removal.4,88

Focal Ischemic Stroke Models

Various ischemic stroke models have been developed during the past 30 years.89 However, the main models in use today are the unilateral middle cerebral artery occlusion (MCAO) models employed in either rats or mice. Since these models were first developed in the 1980s, the MCAO has been variably induced by surgical ligation or cauterization via a small craniotomy over the MCA, passage of a intraluminal nylon suture up into the ipsilateral cerebral circulation via the external carotid in the neck, or via injection of a small autologous thrombus into the common carotid artery. The latter two are the most commonly employed today, and the thromboembolic paradigm is the most clinically relevant, since the majority of human focal ischemic strokes involve a thromboembolic occlusion of the MCA. The MCAO models come in two varieties, temporary and permanent. The temporary MCAO involves removal of the vascular occlusion at varying times (30, 60, 90, 120, 180 min) after the onset in order to allow reperfusion of the ischemic tissue to take place. This experimental scenario, which is accomplished by surgical removal of the extraluminal or intraluminal occlusion device, mimics either the instance where spontaneous thrombus dissolution may take place during the first 3 hours after the beginning of the stroke due to the activation of endogenous thrombolytic processes (believed to be a fairly rare occurrence), or the situation where the stroke victim is treated with the thrombolytic agent tissue plasminogen activator (tPA) for the purpose of dissolving the clot and restoring recirculation.

Although removal of the vascular occlusion and re-establishment of the normal cerebral circulation is an obviously desirable therapeutic goal, it is well known to be a two-edged sword that can lead to reperfusion injury, which is caused by a burst of reactive oxygen species in the previously ischemic brain tissue. Thus, there is a need in this situation for a neuroprotective agent to reduce both the pathophysiological events set in motion by the ischemic insult as well as the subsequent deleterious side effects of pharmacological recirculation. Accordingly, the temporary MCAO models are most useful for evaluating neuroprotective strategies that may be used in conjunction with tPA and other thrombolytic agents. However, since pharmacological thrombolysis can lead to secondary cerebral hemorrhage if used beyond the first few hours, it is only able to be safely employed in patients who are available for emergency treatment during the first few hours after the onset of their strokes. This is only feasible in a small fraction of ischemic stroke patients. Furthermore, MCAO animal studies have shown that reperfusion beyond the first 3 hours is unable to lessen the extent of ischemic damage. Thus, the temporary MCAO models, although widely used in stroke research, actually have limited relevance to the majority of MCA territory strokes.

The second variety of focal ischemic stroke model, the permanent MCAO, where the occlusion is permanently left in place, is therefore a better model of the vast majority of strokes where recirculation has not been re-established either spontaneously or pharmacologically during the critical first few hours after stroke onset. In this instance, the therapeutic goal is simply to try to reduce the expansion of the ischemic damage from the severely ischemic “core” area, which is doomed to infarction if reperfusion does not occur during the first 3 hours, into the surrounding “penumbral” area. The ischemic penumbra is potentially salvageable for several hours due to its partial circulation from collateral blood vessels. In this author’s opinion, the permanent MCAO version is the best option for preclinical evaluation of potential neuroprotective agents since it is more relevant to the overall focal ischemic stroke population where early reperfusion is not all that common. However, the testing of compounds in the temporary MCAO paradigm is probably more relevant to post-TBI ischemic insults caused by subdural or epidural hematomas, since their surgical removal leading to reperfusion of the previously compressed ischemic tissue underlying the hematoma is generally attempted as soon as possible.

Most stroke research with either the temporary or permanent MCAO models is carried out in mice or rats. The primary endpoints are histological demonstration of a reduction in brain infarct size and improvement in motor function typically determined between 24 to 72 hours or sometimes as long as 1 week after stroke onset. However, MCAO models have been developed and are occasionally used in higher species including the cat, monkey, and baboon. Obviously, the use of these for neuro-protective drug evaluation carries considerable expense.

Some investigators, a minority, believe that it is important to replicate pharmacological neuroprotective actions in these gyrencephalic species prior to movement of the compound into human clinical trials. In actuality, there is no solid comparative evidence that supports the notion that neuroprotective effects seen in rodent stroke models may not be predictive of human efficacy. Furthermore, there is presently no firm data that supports the commonly held idea that the therapeutic time window for a particular neuroprotective mechanism in a rat stroke model (e.g., 1 hour) may be longer in nonhuman primates or humans (e.g., 6 hours). On the contrary, the fact that various neuroprotective compounds that demonstrated a rather limited (1–2 hours) therapeutic window for reduction of infarct size in rat MCAO models, subsequently failed to improve outcome of stroke patients in clinical trials where the treatment initiation time varied from 6 to 24 hours, and is consistent with the concept that the therapeutic window for neuroprotective effects may not be all that different between rodents and primates. At least, no difference has been firmly demonstrated.

ISSUES THAT NEED TO BE ADDRESSED IN PRECLINICAL NEUROPROTECTIVE DRUG EVALUATION

Last, since the main purpose of TBI modeling is to enable the discovery of neuroprotective approaches that are mostly pharmacological, this chapter closes with the following list of issues or questions that need to be addressed during the preclinical evaluation of drugs for acute neuroprotection.

  • 1. A thorough demonstration of the time course of the target pathophysiological mechanism in relevant animal models. This is needed to determine when treatment needs to begin and how long it must be maintained. This should be done in both male and female animals based upon several studies showing that the time course and magnitude of post-traumatic pathophysiology and neurodegeneration may differ greatly between genders in certain models.53,90,91
  • 2. A rigorous dose-response analysis in regard to effects on the target mechanism, ability to reduce post-traumatic neurodegeneration, and improve behavioral recovery.
  • 3. A correlation of the neuroprotective action with plasma and CNS tissue pharmacokinetics; that is, a definition of the effective neuroprotective concentration and a dosing protocol that is adequate to maintain the therapeutic concentration for as long as the target secondary injury mechanism is active.
  • 4. A comparison of single versus multiple dose regimens in order to establish an optimum treatment regimen (i.v., bolus plus infusion makes the most sense).
  • 5. A determination of the therapeutic window in order to know how early treatment must begin. It has been argued that even if a particular agent only has an hour window in a rat stroke, TBI, or SCI model, the window in humans with the corresponding condition is likely to be much longer. However, there is little evidence to support this assumption. Consequently, clinical trial design should probably take the preclinical therapeutic window definition for a particular agent seriously in regard to how soon the compound may need to be given to patients. With this in mind, a failure to demonstrate a clinically practical therapeutic window for a particular agent in an animal model may mean that this agent and its corresponding secondary injury mechanism may be too short to be effectively addressed in real-world therapeutics.
  • 6. The above parameters’ dose-response, optimum treatment duration, and therapeutic window are most likely to vary between TBI, ischemic stroke, cardiac arrest/resuscitation, SAH, and SCI models.
  • 7. A comparison of the neuroprotective pharmacology (e.g., dose-response, optimum treatment duration and therapeutic window) in multiple injury models (e.g., focal vs. diffuse TBI) in order to determine whether the agent in question only works in certain types of injuries.
  • 8. A comparison of the neuroprotective pharmacology in male versus female animals.
  • 9. A determination of pharmacodynamic and pharmacokinetic interactions with other commonly used ancillary treatments (e.g., anticonvulsants, minor and major tranquilizers).

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Figures

FIGURE 11.1. Heterogeneity of severe traumatic brain injury (TBI).

FIGURE 11.1

Heterogeneity of severe traumatic brain injury (TBI). Computed tomography (CT) scans of six different patients with severe TBI, defined as a Glasgow Coma Scale score of <8, highlighting the significant heterogeneity of pathological findings. CT scans represent patients with epidural hematomas (EDH), contusions and parenchymal hematomas (contusion/hematoma), diffuse axonal injury (DAI), subdural hematoma (SDH), subarachnoid hemorrhage and intraventricular hemorrhage (SAH/IVH), and diffuse brain swelling (diffuse swelling). (Reproduced with permission from K.E. Saatman et al., J. Neurotrauma 25: 719–738, 2008.)

Tables

TABLE 11.1

Reasons for Past Failures in Neuroprotective Drug Discovery and Development

Inadequate Understanding of Secondary Injury Mechanisms
Lack of definition of time course of glutamate receptor functional changes
Lack of definition of the sources and spatial and temporal characteristics of reactive oxygen generation; inability to rationally determine therapeutic window and optimum treatment duration
Lack of understanding of the interrelationship of secondary injury mechanisms
Focus on secondary injury mechanisms with short therapeutic windows; need to identify and target injury mechanisms with longer therapeutic windows
Lack of understanding of the relative therapeutic windows in animal models and humans; is the time course of secondary injury in mice, rats, and men similar
Inadequate Preclinical Testing
Lack of testing in multiple models
Failure to compare efficacy in male and female animals
Incomplete dose-response and definition of therapeutic plasma levels
Incomplete definition of therapeutic window
Lack of definition of pharmacokinetics, timing of needed maintenance dosing, and optimum treatment duration
Poor Clinical Trial Design
Gross mismatch between preclinical and clinical testing
Imprecise/insensitive outcome scales (e.g., Glasgow Outcome Scale)
Lumping of all kinds of TBIs
Lack of identification and a priori plan to analyze pathophysiological subgroups (e.g., tSAH)
Lack of biomarker to follow the progression of the pathophysiology and monitor mechanistic drug effects
Lack of standardization of acute TBI treatment guidelines and neurorehabilitation protocols between sites

TABLE 11.2

Injury Types Seen in 17 Individual TBI Patients

PatientM/FPostresuscitation CCSSAHDAIEDHSDHCortical ContusionIPHIVHSkull Fractures
1F10✓
2M9✓✓
3M9✓✓
4M6✓
5M6✓
6F6✓✓✓
7F7✓✓
8M8✓✓
9F6✓✓✓
10M6✓✓✓
11M7✓✓
12M4✓
13M7✓✓✓
14M6✓
15M7✓✓
16M9✓✓✓
17M5✓✓

Note: Patients presented at the University of Kentucky Medical Center between June 2010 and November 2012. Thirteen patients ate (GCS = 9–10). DAI = diffuse axonal injury; EDH = epidural hematoma; IPH = intraparenchymal hemorrhage; IVH = or female; SAH = traumatic subarachnoid hemorrhage; SDH = subdural hematoma.

TABLE 11.3

Summary of TBI Characteristics Seen in 17 Moderate or Severe TBI Patients

Patient Characteristics (N = 17)Values (Range)
Age (Years)32 (18–63)
Male (%)77%
Postresuscitation GCS7(4–10)
Initial Intracranial pressure (ICP; mm Hg)13.6 (2–23)
Injury TypeN (%)
Traumatic subarachnoid hemorrhage (SAH)7 (41.2%)
Diffuse axonal injury (DAI)5 (29.4%)
Epidural hematoma (EDH)2 (11.8%)
Subdural hematoma (SDH)4 (23.5%)
Cortical contusion5 (29.4%)
Intraparenchymal hemorrhage (IPH)5 (29.4%)
Intraventricular hemorrhage (IVH)2 (11.8%)
Skull fracture4 (23.5%)
Extracranial injury6 (35.3%)

Note: Patients were admitted to the University of Kentucky Medical Center between June 2010 and November 2012.

TABLE 11.4

In Vivo TBI or TBI Relevant Models Employable for Discovery of Neuroprotective and Neurorestorative Agents

Traumatic Brain Injury Models
Diffuse TBI
Rat or mouse central fluid percussion
Rat or mouse impact acceleration
Rat or mouse closed skull weight drop, single or repetitive
Pig, cat, rabbit, sheep, or nonhuman primate inertial acceleration (nonimpact)
Focal TBI
Rat or mouse controlled cortical impact (CCI)
Rat open skull weight drop Rat bifrontal contusion
Mixed Diffuse/Focal
Rat or mouse lateral fluid percussion
Complex
Rat or mouse diffuse of focal complicated by secondary insult (hypotensive, hypoxic, or traumatic insult)
Penetrating
Rat penetrating ballistic-like brain injury (PBBI)
Blast
Rat blast tube
Traumatic Brain Injury Relevant Models
Subarachnoid Hemorrhage
Rabbit, cat, or dog intracisterna magna injection of autologous blood
Rat intracranial injection of autologous blood via dorsolateral cranial burr hole
Monkey SAH via surgical placement of autologous blood clot around base of MCA
Intracerebral/Intraparenchymal Hemorrhage
Rat striatal ICH
Subdural Hematoma
Rat intracranial injection of autologous blood via dorsolateral cranial burr hole (same as SAH model)
Focal Ischemia
Rat or mouse temporary middle cerebral artery occlusion (MCAO)-microclip or intraluminal suture for 30 min–2 hours
Rat or mouse permanent MCAO-electrocoagulation or intraluminal suture
© 2016 by Taylor & Francis Group, LLC.
Bookshelf ID: NBK326712PMID: 26583171
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