Chronic Histopathological and Behavioral Outcomes of Experimental Traumatic Brain Injury in Adult Male Animals

Overview and purpose

It is well-established that traumatic brain injury (TBI) leads to diverse histopathological and behavioral consequences that begin in the acute period (hours to days) and persist chronically (weeks, months, and years after injury). Chronic symptoms negatively affect survivors' quality of life and hinder their independence and ability to return to preinjury responsibilities. ^{1 –6} Even with modern medical care, an estimated 3.2–5.3 million Americans are living with one or more residual problem attributed to TBI. ^2,7 There is an impetus to better understand the long-term consequences of TBI, given that chronic symptoms are distressing for both TBI survivors and their families and are coupled with significant health service utilization and cost.

Animal models have been a mainstay of TBI research for over a century. ^{8 –11} Several types of experimental TBI models have been developed to model the consequences of TBI, and four of the most commonly used options are discussed in this article: fluid percussion injury (FPI); controlled cortical impact (CCI); blast TBI (bTBI); and closed-head injury (CHI). In this review, separate headings are used for different injury induction methods with the test species, location, or injury severity denoted in the body of the text when relevant. It is important that additional long-term studies of experimental TBI are conducted so that the persistence and progression of TBI pathology may be better understood, given that this information can be applied to guide the development of therapeutic interventions that reduce chronic disability. To date, the chronic consequences of experimental TBI remain less characterized than the acute pathology. This was confirmed by Gold and colleagues, who reviewed 314 data-based publications from experimental TBI studies, which met the following criteria: used rodent models, compared TBI-exposed cases to controls, and included functional outcome assessments. ¹² The researchers found that only 32% of all experimental TBI publications meeting the above-mentioned criteria included outcomes beyond 1 month postinjury, and a mere 8.6% of studies assessed outcomes beyond 2 months postinjury. ¹² Other TBI researchers have acknowledged that only limited attention has been given to understanding the chronic effects of experimental TBI. ¹³ Only a handful of studies have included postinjury outcomes out to 1 year or beyond. ^{14 –18} Despite the limited quantity of evidence, the literature that is available from experimental models of TBI suggests that histopathological and functional outcome deficits persist into the long term, as observed in TBI survivors. ^{19 –21}

The purpose of this review is to summarize the key evidence available from long-term studies of FPI, CCI, bTBI, and CHI. Less commonly used methods for inducing experimental TBI, such as cold probe injury, ²² penetrating ballistic-like brain injury, ^{23 –26} and combinatorial models (e.g., blast+stress), are not included. In this review, long-term outcomes, defined as evidence collected at or beyond 2 weeks after injury, are emphasized. Our rationale for using a 2-week cutoff for chronic outcomes in this review is based on a desire to provide the most comprehensive review possible, keeping in mind the relative dearth of long-term outcome assessments highlighted in the Gold and colleagues review. Notably, there is no consensus regarding how the time course of recovery observed in humans compares to those observed in the various experimental TBI models.

The resources at a large university library system and online search engines (e.g., PubMed, PubMed Central, Google Scholar, and Web of Science) were used to identify relevant literature. In total, 193 articles were identified, as summarized in Table 1. Assessment time points are not always reported and when they are the units of time varied (e.g., days, weeks, months, or years). To facilitate ease of comparison in this review, all data collection time points were converted to approximate number of days based off a 30-day month and a 365-day year. Study length in days was used to create ordinal groups: 14–29, 30–59, 60–89, 90–119, 120–149, 150–179, 180–209, 210–239, 240–269, 270–299, 300–329, 330–365, and 366+days postinjury. Group frequencies were generated, which revealed that, of the long-term studies identified, only one quarter (26.4%) collected outcomes at or beyond 2 months (60 days) postinjury. A mere nine studies (4.6%) collected outcomes at 1 year (365 days) postinjury, ^{14 –18,27 –30} with one extending beyond 1 year (0.5%). ^26,31

Of the 193 articles identified, the vast majority (96.4%) used rodent models, with 65.3% of studies being rat studies and 31.1% mouse studies. The remaining 3.6% of studies used pigs, nonhuman primates, and rabbits (Table 1). No exclusions were made based on the species used, but efforts were made to denote the species with the evidence, especially for nonrodent models.

Notably, the vast majority of long-term studies identified used male animals exclusively (83.4%), whereas only a handful of studies used females exclusively (4.1%) or both males and females (3.6%). The remaining 8.8% did not specify the sex of the animal used (Table 1). In this review, the long-term evidence highlighted will be limited to studies exclusively and explicitly using male animals. The rationale for this decision is 2-fold: 1) outcomes of TBI may be moderated by sex and/or the effect of sex hormones ^{32 –35} and 2) human males in all age groups are more likely to experience TBI than females. ³⁶ Whereas long-term evidence will be limited to males, studies using female animals or not specifying animal sex may be cited in supporting text (e.g., introducing/defining concepts, describing models, in the Discussion section).

Age of the test animals is another study variable that is often not reported by researchers. Indeed, nearly one third (31.6%) of studies did not specify the age, with many of these also not indicating weight, which could be used to approximate age of the test animals. Another one quarter of the studies (25.9%) vaguely listed the age as adult. A mere 12 studies (6.2%) explicitly described the sample as consisting of immature (i.e., juvenile, pediatric, and adolescent) animals or provided animal ages that correspond to an immature animal of that type/strain. These 12 studies were excluded from the evidence highlighted in this review. However, owing to the large percentage of studies that did not specify animal age, studies were not excluded from this review if age was unspecified, as was true for studies that did not specify sex.

For each of the five TBI models included, a brief historical overview is provided with commonly used species noted. A summary of key evidence from studies using each model is divided into histopathological and functional outcomes. The histopathological consequences of interest in this review are gross morphological changes, ventricular enlargement, gray/white matter (GM/WM) shrinkage, cell death, axonal injury, cerebrovascular histopathology, inflammation, and neurogenesis. With respect to functional outcomes, this review includes overall neurological function, motor function, frontal lobe function, and stress-related outcomes.

In the summary of the evidence provided, injury severity is sometimes included to provide additional details that may be of interest to readers. However, it is important to note that injury severities included in this review are based off the original description of injury level from the primary source; these severity levels may not be consistent across research groups complicating comparisons across studies. Moreover, these injury severity levels may or may not correlate with clinical TBI severity.

In summarizing the evidence pertaining to each outcome of interest for the five models, the emphasis is on evidence gathered over the longest study period from the publications identified during the literature review. After the model-specific discussion of the long-term histopathological and behavioral consequences of experimental TBI, a brief description of the comparisons and contrasts between the models is provided, including a table to summarize key comparisons. The primary aims of this review are to highlight the literature detailing the chronic histopathological and behavioral outcomes of TBI, and leave the reader with an understanding of the techniques available for the study of chronic TBI in animals. Careful study planning is important to select the appropriate model characteristics to best address the research question(s).

Outcomes of interest

A short definition/description of each histopathological and behavioral outcome is provided, and the clinical relevance of the outcomes of interest is noted. Gross morphological and histopathological changes are apparent with the use of simple staining and microscopy. Examples include gross contusion, lesion, or volume loss of particular brain structures. These overt changes in brain morphology have been observed after clinical TBI ³⁷ and replicated in experimental models. ³⁸ The manifestation of post-traumatic functional impairments can exist in the absence of overt morphological changes, ³⁹ and thus conclusions from studies are strengthened by the inclusion of both histopathological and functional deficits.

Three related changes are discussed under a single heading: ventricular enlargement, shrinkage of GM, and shrinkage of WM. Ventricular enlargement has been observed after clinical TBI where it correlated with deficits in cognition and memory. ^{40 –43} Similarly, GM shrinkage has been observed after clinical TBI. ^44,45 Gale and colleagues used voxel-based morphometry in TBI survivors approximately 1 year postinjury and reported that GM concentration was significantly lower than in matched controls. ⁴⁵ WM atrophy contributes to tissue shrinkage observed after clinical TBI ^42,46 and can be studied using animal models. ^28,47 Changes in GM/WM concentration ^28,29,48,49 and ventricular enlargement have also been reported after experimental TBI. ^14,27

A factor contributing to the aforementioned changes to GM and WM is cell death. Cell death occurs in multiple types of central nervous system (CNS) cells after TBI through multiple mechanisms: apoptosis, necrosis, and autophagy. ^50,51 Depending on the types of cell death (or subset of a particular cell death pathway) that are of interest, the researcher can choose between available models (e.g. FPI, CCI, bTBI, and CHI), which differ in their histopathological and behavioral consequences, as described in this review. Discussion of cell death will be broken down under three headings pertaining to apoptosis, necrosis, and autophagy; when relevant, a discussion of the patterns of cell death will be included.

Early evidence of axonal injury, a common TBI pathology, was reported nearly 70 years ago by Rand and Courville, who observed histopathological changes throughout many brain regions post-TBI. ⁵² Additional evidence accrued regarding axonal pathology at early post-TBI time points in varying injury severities. ^{53 –55} More in-depth categorization of these changes occurred during the 1980s, leading to the now widely use concept of diffuse axonal injury (DAI). ^56,57 DAI is characterized by early axonal changes and swelling during the first 1–2 days post-TBI with the progression to more extensive secondary damage and ultimate axonal disconnection. ⁵⁸ Axonal injury has been reported in numerous experimental TBI models. ^57,59,60

Cerebrovascular histopathology includes disruption of the blood–brain barrier (BBB), endothelial cell damage, and dysregulation in cerebrovascular blood flow. These changes to the cerebrovascular system have been observed in the clinical setting post-TBI. ^{61 –64} Evidence of cerebrovascular histopathology has also been reported in experimental models of TBI. ^{65 –67}

The study of inflammation began in antiquity, with Celsus. ⁶⁸ More recently, scientific understanding of inflammation has broadened to include both the multiple influences and outcomes when defined as a “multi-mediated phenomenon, of a pattern type in which all mediators would come and go at the appropriate moment…increasing vascular permeability, attracting leucocytes, producing pain, local edema and necrosis.” ⁶⁹ Historically, the brain was considered immune privileged; however, CNS inflammation has since been discovered, as evidenced by major histocompatibility complex expression, glial activation, acute phase response, complement activation, expression of adhesion molecules, inflammatory mediator synthesis, edema, and immune cell invasion. ⁷⁰ In the context of clinical TBI, inflammation represents a key pathophysiological process during both the acute and chronic periods ^{46,71 –73} and is replicated by experimental TBI. ^49,74,75 Whereas microglial and astrocytic responses are also part of the inflammatory cascade, they are not emphasized in this review; rather, the focus is primarily on cytokines because these are better categorized in the long term postinjury.

Neurogenesis can be defined as the process in which new neurons are generated through differentiation and maturation of neural stem cells and neuronal precursor cells. ^{76 –79} Following the observation that ongoing neurogenesis occurs in the adult human brain, ^80,81 this physiological process has been explored in animal models as a mechanism by which new neurons could be generated to compensate for cell loss after TBI. ^{82 –86} Whereas adult neurogenesis occurs in both the subgranular zone of the hippocampus and the subventricular zone of the lateral ventricle, this review focuses on hippocampal neurogenesis because the hippocampus is an important contributor to specific cognitive tasks. ^{87 –90} Studies using many of the aforementioned animal models of TBI demonstrate that therapeutic interventions designed to enhanced neurogenesis may contribute to improved neurobehavioral function post-TBI. ^{84,91 –93}

There are six main behavioral outcomes of interest in this review; each outcome is studied using a variety of experimental methodologies. Overall neurological function is negatively impacted by clinical TBI and can be evaluated in TBI patients using tools such as the Glasgow Outcomes Scale (GOS), Glasgow Outcomes Scale Extended, and Neurobehavioral Rating Scale. ^{94 –98} In experimental models of TBI, overall neurological function is assessed using various composite tools. ^{99 –102}

Motor deficits are common after clinical and experimental TBI. In pre-clinical studies, motor deficits are assessed using the beam balance task (BBT), beam walking task (BWT), inclined plane task, cylinder test, foot fault test, bilateral tactile adhesive removal task, rotarod, and rotating pole task. Impairments in acute and chronic motor function have been observed on these assessments when applied to studies using varied experimental TBI models. A composite neuroscore, which combines several motor assessments into a single score, ^99,103 can be used by researchers to evaluate gross and/or fine motor function, depending on the task(s) utilized and study time frame.

Cognitive dysfunction is cited by TBI survivors as one of the greatest contributors to reduced quality of life. ¹⁰⁴ In animal models, the components of cognitive function commonly studied include spatial memory/learning, nonspatial learning, and reference memory. Spatial memory is most commonly assessed using the Morris water maze (MWM) hidden platform task, reference memory with the MWM probe trial, and nonspatial learning using the passive avoidance task. ¹⁰⁵ Passive learning is assessed using cued and contextual fear conditioning. ¹⁰⁶

Frontal lobe function encompasses executive function, memory, language, aggression, and reasoning. ^107,108 TBI in humans is known to affect the frontal lobe and cause dysfunction in the cognitive processes controlled by this brain region. ^{109 –111} However, frontal lobe dysfunction remains understudied in animals because it is challenging to propose experimental analogs of the complex frontal lobe functions observed in humans (e.g., executive function, impulse control, and language). To date, frontal lobe function after experimental TBI has been most studied in the context of CCI.

Several stress-related outcomes have been associated with TBI, most notably post-traumatic stress disorder (PTSD). ^{112 –117} Fear and stress can lead to emotional and physical consequences, including outcomes associated with sympathetic nervous system actions. ^118,119 In experimental models of TBI, heightened stress and fear have been reported. ^120,121 Often, bTBI studies include stress-, fear-, and anxiety-related outcome measurements, such as the elevated plus maze, zero maze, and responses to predator stress exposure. ^122,123

Shrinkage of gray/white matter and enlargement of ventricles

Carbonell and Grady reported that parasagittal FPI in mice led to WM degeneration in multiple brain regions, which was evident by 4 days postinjury and progressed at least 35 days postinjury. The researchers concluded that the spatial pattern of damage was similar in mice and rats post-FPI, but did note one key difference: Mice exhibited a shorter period of damage, particularly within the thalamus and hippocampus. ¹³⁷ In rats, enlargement of the lateral ventricles has been reported 8 weeks post-FPI. ¹³⁸ Long-term ventricular enlargement post-FPI has also been reported by Nonaka and colleagues, who found progressive ventricular expansion up to 1 year after parasagittal FPI in rats. ¹⁶

Cell death

Axonal injury

The FPI model has been used to characterize the progression of axonal injury. ^59,146 However, only a handful of studies have evaluated chronic axonal injury. A study by Pierce and colleagues was among the first to include both histopathological and behavioral outcomes out to 1 year after severe lateral FPI. ²⁷ Notable findings from this study were year-long functional deficits (e.g., learning, motor), which co-occurred with axonal degeneration in the injured cortex, corpus callosum (CC), and striatum. Smith and colleagues published several experimental TBI studies that contributed to the understanding of the prolonged time course of traumatic axonal damage. ^{147 –150} Axon swelling throughout the brain has been observed out to 6 months postinjury, and time-dependent reductions in the number of myelinated axons in the thalamus and cortex were reported up to 1 year postinjury. ²⁹ In a study of parasagittal FPI, Dietrich and colleagues found a spatial relationship between areas where axonal damage was found and those where long-term GFAP messenger RNA was observed. ¹⁵¹

Using an FPI model to produce a mild TBI (mTBI), Spain and colleagues found evidence of spatially progressive axonal degeneration within the external capsule and the dorsal thalamus up to 6 weeks after insult. At the 4-week time point, elevated levels of axonal bulbs within the external capsule were evident in injured animals, whereas no major differences were present in other areas of the brain. In addition, myelin basic protein immunostaining showed no damage to myelin during any time point within the study. ¹⁵²

Cerebrovascular histopathology

Cerebrovascular dysfunction has not been well studied during the chronic period post-FPI. One study by Guo and colleagues documented evidence of ongoing angiogenesis out to 2 weeks postinjury. In this study, CD34-positive cells were reported starting 72 h after injury and persisting for 14 days post-TBI. ¹⁵³ Existing evidence suggests that CD34-positive cells are directly incorporated into expanding vascular cells. ¹⁵⁴ CD34-positive cells have also been found to impact secretion of angiogenic growth factors, such as vascular endothelial growth factor (VEGF). ¹⁵⁵ Thus, CD34-positive cells are believed to promote angiogenesis both directly and indirectly. However, the exact mechanisms by which CD34-positive cells contribute to angiogenesis remains unknown. ¹⁵⁶

Neurogenesis

A recent experimental study observed neurons expressing neural progenitor and immature neuron markers within perilesional regions, including the cortex, WM, and hippocampus, in resected tissue from adults humans post-TBI. ¹⁵⁷ After an FPI, the hippocampus exhibits a robust increase in cellular proliferation of neural stem cells in the subgranular zone of the dentate gyrus within the first week of brain injury ^86,158,159 ; however, the transient elevation in the rate of cellular proliferation returns to baseline by 2 weeks postinjury. ⁸⁶ Cellular proliferation is identified by the incorporation of bromodeoxyuridine or other thymidine analog into the DNA during cellular replication. As an alternative, researchers can assess cell proliferation using Ki67 immunoreactivity; Ki67 is a protein expressed during several phases of the cell cycle (e.g., late G₁, S, G₂, and mitosis). ^160,161 Many of the newly proliferated cells will adopt an astrocytic or GFAP-positive lineage, but a subpopulation will differentiate and express neuronal markers in the weeks postinjury. ¹⁶² In the context of FPI, two studies demonstrate that newly generated neurons extend axonal projections that integrate into CA3 at 2 ¹⁵⁹ and 8 weeks ¹⁶² postinjury. These studies demonstrate that FPI stimulates a post-traumatic neurogenic response that results in an elevation in the number of new neurons that can functionally incorporate into the hippocampus in the weeks after injury.

Overall neurological function

Kabadi and colleagues found distinct injury-induced neurological score impairment after sham, mild FPI, and moderate FPI out to 3 weeks postinjury, ¹³¹ with scoring as previously described. ^{163 –165} Briefly, neurological score was calculated based on four motor assessments: 1) right (R) and left (L) forelimb contraflexion when rats were suspended by the tail; 2) R&L hind limb flexion when rats were suspended by the tail; 3) resistance to R&L lateral pulsion; and 4) ability to stand on an inclined angle board. ¹³¹ Another study found that severe lateral FPI worsened composite neuroscore performance out to 2 months postinjury. ²⁷ McIntosh and colleagues evaluated composite neuroscore score using five motor assessments: 1) forelimb flexion during tail suspension; 2) circling behavior during spontaneous ambulation; 3) decreased resistance to lateral pulsion; 4) ability to balance on a narrow (2 cm wide) wooden beam; and 5) ability to stand on an angled board with the maximal angle the animal could stand on for 5 sec recorded. ⁹⁹ This 5-point neurological assessment results in scores ranging from 0 to 20, interpreted as follows: 20=normal, 15=slight impairment, 10=moderate impairment, 5=severe impairment, and 0=afunctional. Whereas McIntosh and colleagues found no effects of mild injury, moderate FPI led to deficits up to 4 weeks postinjury in this study. ¹⁰³ Other researchers have reported neuroscore deficits post-FPI, as described in the following section.

Motor function

Deficits on the BBT have been observed up to 4 months post- FPI. ^166,167 Impaired rotarod performance beyond 4 weeks postinjury has been observed after lateral FPI. ¹⁶⁸ A number of motor behaviors assessed by Smith and colleagues, including BWT, skilled forelimb reaching, and locomotor placing, were assessed after lateral FPI. ¹⁶⁹ The BWT is commonly used by researchers to study the consequences of experimental TBI on fine motor performance. In this assessment, animals are motivated, using negative reinforcement, to traverse a narrow beam. Deficits on the BWT have been observed after a single head trauma modeled using lateral FPI, ^{167,170 –172} as well as repeated head trauma modeled using lateral FPI. The bilateral adhesive removal task is another fine motor task that has shown deficits out to 1 month after lateral FPI. ¹⁷³ Other less-common assessments that have shown long-term deficits in FPI studies include the foot fault test of locomotor function, ¹³⁰ the rotarod test of balance, ¹⁷³ the cylinder test of dorsal raphe nuclei and median raphe function, ¹⁷⁴ and the rotating pole test of coordination, ^166,168,173 all of which have shown deficits 1 month postinjury.

Other common assessments of motor function are the composite neuroscore and MWM. Early use of the composite neuroscore by McIntosh and colleagues combined five motor assessments into a single test, as described above. ^99,103 Motor deficits assessed using the composite neuroscore have been reported at 1 month postinjury in several studies. ^{27,166,168,175 –177} One of the studies found that deficits persisted out to 2 months postinjury. ²⁷ The MWM, discussed below in relation to its use for assessment of cognitive function, such as spatial learning/memory and reference memory, also collects swim speed data that provides information about overall motor function. Long-term deficits on swim speed have been observed after both isolated lateral FPI ¹⁷⁸ and repeated lateral FPI. ¹⁷⁹

Cognitive function

Learning deficits assessed using the MWM were evident out to 2 weeks in a rat model of severe FPI. ¹⁸⁰ MWM deficits have been observed to persist for up to 4 weeks post-FPI in some studies ^179,181,182 and 8 weeks postinjury in others. ^167,183,184 When the severity of injury was increased, MWM deficits were observed out to 1 year postinjury. ²⁷ An alternative assessment of learning, the reversal learning test, also showed impairments 8 weeks after a single lateral FPI ¹⁸⁶ or repeated lateral FPI. ^179,181

Reference memory is also impaired after FPI. Injury effects on the MWM have been reported out to 2 weeks post-FPI. ¹⁸⁵ Other studies in rodents have reported reference memory deficits out to 1 month after both lateral FPI, ^{130,175,176,184,186,187} as well as medial FPI. ^188,189 Sun and colleagues observed cognitive deficits assessed by the MWM on days 11–15 and 26–30 postinjury; in this study, deficits were resolved by days 56–60 postinjury. ¹⁶²

Radial arm maze task for assessment of spatial learning and memory was found to be sensitive to FPI out to 7 weeks postinjury. ¹⁶⁷ As an alternative, researchers can assess spatial learning/memory using the Barnes Maze, where rodents are placed on a well-lit circular platform with 40 holes and trained to locate a specific hole under which a goal box is found. ¹⁹⁰ Injured animals assessed at 1 and 3 months postinjury impaired Barnes Maze performance. ¹⁹¹ Impaired spatial learning was observed 3 weeks post-FPI with concomitant localized axonal damage within the external capsule and dorsal thalamus, despite unaltered myelin. ¹⁵²

Stress-related outcomes

Overall, stress-related outcomes are rarely assessed into the long term post-FPI. Anxiety, assessed using the elevated plus maze, has been reported out to 1 month after a single lateral FPI, ¹⁷² as well as after repeated lateral FPI. ^179,181 Depression-like behavior in rodents are observed 1 month after lateral FPI in the forced swim test. ¹⁷⁹

Gross morphological and histological changes

One study compared animals exposed to CCI and sham and found that CCI-exposed animals had vastly larger contusion volumes at 3 weeks postinjury. ¹⁵ Another notable finding of this study was that the size of the contusion nearly doubled by 1 year post-CCI. ¹⁵ Though progressive gross changes have been reported, evidence of long-term morphological changes remains sparse.

Enlargement of ventricles and shrinkage of gray/white matter

Post-CCI in rats, volume of ipsilateral ventricles increased approximately 3-fold between 3 weeks and 1 year postinjury, suggesting chronic loss of GM and WM. ¹⁵ A CCI model of repetitive mTBI found that bilateral contusion injuries spaced 1 week apart resulted in continued damage to the myelin within the CC that was not present in animals with only a single injury at 60 days postinjury. ¹⁹⁵

Cell death

Axonal injury

Axonal injury has traditionally been included as a short-term outcome post-CCI. ^201,202 Myelin loss is another change to axons that has been reported in the long term post-CCI. Glushakova and colleagues used Luxol fast blue to detect loss of myelin out to 3 months postinjury in conjunction with microbleeds. ²⁰³ The vascular consequences observed in this study are described in additional detail in the next section. ²⁰³

Cerebrovascular histopathology

In the aforementioned Glushkova and colleagues study, microbleeds were associated with myelin loss and glial scarring. Taken together, the findings suggest that vascular damage is associated with the combination of breakdown of the BBB and inflammatory processes. ²⁰³ Moreover, the researchers assert that enhanced understanding of delayed microvascular changes could lead to a better understanding of long-term TBI pathology.

Inflammation

Overall, cyclooxygenase 2 (COX-2) activity produces benefits in the context of TBI; however, ongoing production of COX-2 may be associated with secondary damage from oxidative stress, deficits in cellular metabolism, vascular changes, and associated symptoms. ²⁰⁴ Evidence of long-term inflammation was reported in a study by Briones and colleagues, in which rats sacrificed one month post-CCI exhibited elevated interleukin (IL)-1β and tumor necrosis factor alpha in both the cortex and hippocampus. ²⁰⁵ The anti-inflammatory cytokine, IL-10, was elevated in both the cortex and hippocampus when assessed one month post-CCI. ²⁰⁵

Neurogenesis

In addition to mature granular neuron loss, immature hippocampal neurons are particularly vulnerable to TBI, resulting in a reduction in immature neuron density up to 2 weeks post-CCI. ^{82,83,206,207} Similar to FPI, CCI stimulates an increase in cellular proliferation and neural stem cell expansion during the week postinjury, ^{82,206,208,209} resulting in the generation of newborn neurons in the granular layer. ^{82,206 –208,210} However, the majority of newly proliferated cells adopt a GFAP-positive lineage, which could include glial cells and neural progenitor pool repopulation in the subgranular zone at 1 month post-CCI. ⁸²

Overall neurological function

Several studies have found that CCI leads to global neurological deficits at 1 month postinjury. ^{211 –214} Although there has been some debate about the reliability, validity, and bias of composite scores, ¹² assessments of overall neurological function have been widely applied in CCI and other experimental models of TBI.

Motor function

Motor function deficits have been reported using the BBT out to 1 month postinjury. ²¹⁵ Fine motor deficits have also been observed 1 month after CCI using the BWT. ^{196,212,216,217} Two experimental CCI studies demonstrated motor function deficits with vermicelli test of manual dexterity up to 8 weeks postinjury. ^218,219 Animals assessed with the cylinder test exhibited paw placement preference out to1 month post-CCI. ²²⁰ Balance deficits have been observed on the rotarod test 1 month post-CCI, ²²¹ as well as out to 11 weeks after moderate CCI. ²²² One study found that grid walk performance was impaired at 4 weeks after CCI in mice, and spontaneous forelimb elevation was reduced at 5 months postinjury. ²²³ Reduced forelimb function after moderate bilateral CCI has been reported up to 18 days postinjury. ²²⁴

Cognitive function

Dixon and colleagues reported that moderate CCI produced persistent spatial memory deficits on the MWM up to 1 year postinjury. ¹⁵ More recently MWM deficits have been observed as early as 2 weeks and up to 1 month post-CCI. ^{47,211,212,214 –216,225 –232} Chronic deficits have been observed out to 1 month post-CCI, when reference memory is assessed using the MWM probe trial. ^{15,227,232,233} In mice, evaluation of learning using the MWM has revealed deficits out to 24 days postinjury ^234,235 and working memory deficits out to 4 weeks post-CCI. ¹⁹⁶ Teresita Briones and her colleagues found that the CCI significantly increased latency on the MWM. ²⁰⁵

Fox and colleagues showed impaired performance on the Barnes Maze task up to 24 days postinjury in mice subjected to CCI. ^217,234 Deficits in working memory function have been reported out to 1 month post-CCI versus controls. ²³³ Nonspatial learning shows an injury effect out to 1 month post-CCI when assessed using the passive avoidance task. ²³⁶ Attenuation of CCI-induced cognitive deficits was observed at 3 months postinjury in mice that exercised beginning 5 weeks after trauma. ²³⁷

Frontal lobe function

The attentional set-shifting test (AST) is a test of frontal lobe function in which animals complete a series of perceptual discriminations of increasing difficulties with food as a reward. Animals are required to both form and maintain an attentional set and then shift from stimulus to stimulus. The food rewards are hidden within terra cotta pots filled with distinctive digging material; each pot has a distinctive odor around the rim that animals use to learn the location of the reward. ^{238 –240} Previous applications of the AST include rodent models of attention deficit hyperactivity disorder, ²⁴¹ Parkinson's disease, ²⁴² aging, ²⁴³ and stress. ^238,244,245 Bondi and colleagues first applied the AST to an experimental TBI study in rats and found deficits at 4 weeks post-CCI. In this study, a series of discriminative tasks were used, with difficulty of discrimination increasing progressively and included simple discriminations, compound discriminations, stimulus reversals, as well as intra- and extradimensional shifts. TBI was found to result in AST deficits, with extradimensional set-shifting and stimulus reversal performance worsening with increasing injury severity. ²⁴⁶

Enlargement of ventricles and shrinkage of gray/white matter

As is true for other models of injury, WM damage is a common pathology of bTBI. WM damage has been observed in both pre-clinical and clinical work and is commonly reported in the CC, brainstem, and parasagittal WM. ^{259 –261} Notably, in a rat model of bTBI induced using a shock tube with body shielding, the most prominent histopathological consequence observed 2 weeks postinjury was degeneration of WM within the fiber track of the cerebellum using silver staining. ²⁶²

Cell death

Axonal injury

Among rat models of TBI, axonal injury has been reported post-bTBI. ²⁶² Garman and colleagues evaluated axonal degeneration using amino silver staining and found multi-focal axonal degeneration at all data collection time points after the initial blast-induced TBI, including up to 2 weeks postinjury. ²⁶² The researchers suggest that silver staining is a valuable, yet underutilized, method of analyzing structural outcomes of experimental TBI, such as axonal injury. ²⁶² However, they also acknowledge that because of the limited sensitivity of the silver staining, it is best to confirm axonal injury using morphological staining patterns. ²⁶²

Axonal injury has also been reported in swine up to 2 weeks post-bTBI; specifically, elevated amyloid precursor protein was found, compared to naïve and sham controls, in a study by de Lanerolle and colleagues. ²⁶³ In a study of blast injury with body shielding in rats, scattered axonal spheroids were observed in the deep cerebellar WM and brainstem at 2 weeks postinjury, suggesting DAI. ²⁶² Axonal density changes have also been reported in the cingulum bundle out to 14 days after laser-induced shock-wave exposure (0.5–1.5 J/cm²) in rats. ²⁶⁴

Aside from axonal injury, changes surrounding myelin have been observed post-bTBI. One month after a single high-intensity (200 kPa) blast in macaques, researchers identified axons completely devoid of myelin staining in both transverse and longitudinal sections at 1 month postinjury, suggesting long-term evidence of axonal pathology post-bTBI. ²⁶⁵

Cerebrovascular histopathology

Available evidence suggests that bTBI leads to impairments in cerebrovascular compensatory responses. This can contribute to secondary insults and development of chronic deficits. However, there remains a gap in the knowledge regarding the effects of blast injury on acute and chronic cerebral vascular reactivity. ²⁶⁶ One bTBI study in rats observed vessel tortuosity within the primary visual cortex along with evidence of collagenous remnants at 10 months after three blast injuries induced on 3 consecutive days. ²⁶⁷ H&E staining after 3 consecutive 74.5-kPa exposures revealed hemorrhage in the region of the fimbria, third ventricle, and adjacent to the periventricular nucleus at 10 months after the final bTBI. ²⁶⁷ In a rat model of bTBI induced using a compression-driven shock tube (20.6 psi), Kovesdi and colleagues observed vasculogenesis, as evidenced by elevated VEGF ²⁶⁸ One month post-bTBI induced using trinitrotoluene, increased BBB permeability was observed. ²⁶⁹ Another study found that early elevation of VEGF remained significantly elevated when assessed 2 weeks after overpressure blast injury in pigs wearing body armor. ²⁷⁰

Inflammation

Long-term evidence of inflammation was reported by Cernak and colleagues. In this study, mild (rupture pressure=183±14 kPa) and moderate (213±17 kPa) bTBI led to elevation in macrophage-inhibiting factor related protein 8, a macrophage-related protein. These changes were reported within the brainstem and hippocampus at 14 and 30 days postinjury. ²⁷¹

Cognitive function

Long-term memory deficits have been reported post-bTBI induced using a compression-driven shock tube (20.6 psi) and was ameliorated by environmental enrichment, when compared to standard housing. ²⁶⁸ Spatial memory deficits have been reported out to 1 month with the Y maze task and Novel Object Recognition (NOR) task post-bTBI induced using 500 g of detonated trinitrotoluene. ²⁶⁹ Similarly, blast overpressure exposure in mice has been associated with memory deficits on the NOR task. ²⁷²

In this study by Cernak and colleagues, the active avoidance response task was used to evaluated cognitive function; mild injury was found to result in cognitive deficits out to 21 days postinjury, but these deficits were attenuated by 30 days postinjury. Conversely, moderate bTBI resulted in persistent cognitive deficits out to 30 days postinjury. ²⁷¹

Stress-related outcomes

A review by Graner and colleagues summarizing the applications of functional magnetic resonance imaging (MRI) suggested that bTBI survivors may experience symptoms related to stress and emotional disturbance related to underlying brain change (e.g., activation of the amygdala). ²⁷³ Clinically, there is a challenge in separating out these problems from more-severe psychological consequences, such as PTSD and major depressive disorder, both of which are common in military personnel. ^274,275

In the context of animal models, bTBI led to prolonged psychological dysfunction for months, including enhanced contextual fear conditioning. ¹²³ Elder and colleagues found that post-TBI stress-related traits persisted for many months after injury, including contextual fear conditioning, increased anxiety, and an altered response in a predator scent assay. Another study reported that rats exposed to blast injury exhibited increased levels of anxiety in the elevated plus maze (EPM) at 1 month; however, at 2 months postinjury, there was no difference between injured and sham animals. ¹²² Kovesdi and colleagues found that injured animals displayed increased anxiety on the EPM at 45 days post-bTBI induced using a shock tube (20.6 psi) ²⁷⁶

Other researchers have evaluated potential strategies to target anxiety post-bTBI. Kovesdi and colleagues observed elevated anxiety peaking at 6 weeks post-bTBI induced within a compression-driven shock tube (20.6 psi), which returned to normal by 2 months postinjury. ²⁶⁸ Long-term evidence of depression has also been reported after blast-induced TBI in mice. Specifically, Cernak and colleagues used two- and three-dimensional mapping of exploratory activity, which revealed hypoactivity, preference for corners, and freezing activity consistent with depressive behaviors at 14 and 30 days postinjury. ²⁷¹

Gross morphological and histological changes

In a repeated mTBI weight drop model, Mannix and colleagues subjected mice to repeated CHI over a 9-day period, in an attempt to replicate repeated human concussion in the absence of convulsion or LOC. No gross morphological changes were observed using MRI out to 6 months after final injury. ²⁸² Further, none of the histopathological testing in this study suggested major WM tract disruption. ²⁸²

Axonal injury

Weight drop impact acceleration is well known for resulting in widespread damage to axons and thus may be chosen by researchers interested in axonal injury as an outcome variable. When silver staining was used after repetitive closed-skull TBI in mice, the silver staining intensity was significantly higher at 1, 2, and 3 weeks postinjury than it was at baseline. ²⁸³ In a weight drop model of repeated CHI (7 injuries over 9 days), no axonal pathology was present when assessed 6 months after final injury. ²⁸²

Neurogenesis

In the context of weight drop impact acceleration, little is known about the vulnerability of immature neurons in the hippocampus after injury. Many of the studies evaluating long-term post-traumatic neurogenesis after weight drop impact acceleration do not assess the hippocampus, but instead the subventricular zone, ^84,284 and were not included in this discussion. Bye and colleagues demonstrated that, similar to FPI and CCI, weight drop impact acceleration increases the rate of cellular proliferation during the week postinjury and leads to an increased number of newly generated neurons in the granular layer at 8 weeks postinjury. ⁸⁵

Overall neurological function

Notably, an early pre-clinical assessment of global neurological function was first applied to a rat model of CHI by Shapira and colleagues. ²⁸⁵ This composite score was based off the following five assessments: 1) righting reflex; 2) presence of hemiplegia; 3) presence of hemiparesis; 4) seeking behavior; and 5) ability to exit a large ring (50 cm diameter). ²⁸⁵ Since the original publication of the Neurological Severity Score (NSS), it has been modified to assess specific aspects of motor function that may be impaired post-TBI. For example, additional assessments of reflexes were added by Shohami and colleagues. ²⁸⁶ More recently, the scoring of the NSS was further expanded to a 14-point scale ^287,288 based off assessment of reflexes, along with motor and sensory functions, which was further adapted into the modified NSS (mNSS). ²⁸⁹ The mNSS is also measured on a 14-point scale and includes assessments of reflexes, motor function, balance, and sensory function. ^287,288,290

Motor function

Coordination deficits assessed by rotating pole test have been reported out to 1 month after repeated CHI. This study also observed deficits in balance assessed by the rotarod task. ²⁹¹ Notably, no additional long-term evidence of motor deficit post-CHI was found by us during the literature search.

Cognitive function

MWM deficits have been observed out to 1 month after a single CHI. ²⁹² Another study reported working memory function deficits post-CHI has been reported out to 1 month postinjury. ²⁹³ No other long-term evidence of cognitive deficits post-CHI was identified.

Stress-related outcomes

Depression- and anxiety-like behaviors have been observed after impact accelerated TBI induced using a modified weight drop protocol. ²⁹⁴ In this study, depression-like behavior was observed post-TBI using the open-field exploration task, the EPM task, along with observations of changes in social behavior and hypermotionality. Anxiety-like behavior was assessed using observations of social interaction and the marble-burying tests. ²⁹⁴

Strengths

Overall, the available evidence suggests that animal models of TBI replicate most histopathological and functional outcomes observed clinically. That said, as discussed previously, some models replicate certain aspects of TBI better than others. In addition to the clinical relevance and advantages over in vitro models, in some applications, experimental studies have advantages over clinical trials. For example, pre-clinical studies of TBI are useful for building a preliminary evidence base and controlling confounding variables that could affect study findings. For example, in animal models, diet and environment can be precisely and acurately controlled. ^215,305

An additional strength of experimental models of TBI is that whereas many of the same histopathological outcomes can be assessed in humans, experimental TBI studies facilitate specifically timed data collection in ways that would be neither feasible nor ethical in humans. Experimental TBI studies allow researchers to precisely and accurately control timing of animal sacrifice and collect fresh postmortem tissue for analysis.

Trials of potential TBI therapeutics represent one of the most important applications of experimental TBI models. It is critical that safety and efficacy of a potential therapeutic be established in animals before translating the agent into clinical trials and, ultimately, patient care. Experimental TBI drug studies also facilitate identification of potential adverse treatment effects.

Weaknesses

The published evidence surrounding the long-term histopathological and functional outcomes of experimental TBI is limited in scope and volume. Few studies include outcome assessments beyond 1 month, with even fewer extending out to 3, 6, or 12 months (Table 1). This narrow timeframe of data collection may obscure our understanding of long-term consequences. Additional research assessing outcomes of TBI into the long term is needed. Arguably, the most significant barrier to conducting long-term experimental TBI studies is the high cost associated with animals, housing, veterinary care, food, and the need for specialized facilities. Concern for the ethical treatment of laboratory animals may further contribute to the preference for shorter-term studies.

Although animal research facilitates control of important variables, including genetics, environment, and diet, there are some unique considerations for researchers to take into account when conducting this type of work. Notably, the clinical population is not as homogenous as those observed in experimental studies, with respect to age, genes, and environment. Thus, additional research efforts are needed to promote generalizability of study findings and/or facilitate clinical translation.

A final limitation of using animal models to study the long-term histopathological and functional outcomes of TBI is that the relationship between structural and functional deficits is not one to one. For example, it is possible to have functional deficits with or without the development of a lesion. Morphological changes can be sufficient to produce functional deficits, but are not necessary. The opposite is also true; pathophysiological changes can occur in the absence of functional deficits. For instance, in a weight drop model of focal brain injury in rats, there was inflammation in the absence of functional deficits. ¹³ That said, limitations in the available research tools could prevent consequences of TBI from being detected.

Closing remarks

Animal models of TBI have been widely used to understand the histopathological and behavioral consequences of brain trauma in humans. Animal models of head trauma remain relevant today. Researchers using animal models to study TBI have a number of critical decisions regarding experimental parameters to consider, including, but not limited to, the species to use, injury method, severity, injury location, and outcome measures. As part of the effort to promote rigorous TBI studies, researchers are advised to follow the aforementioned criteria proposed by the NINDS. ²⁹⁵ Researchers considering the use of an experimental TBI model should give careful consideration and attention to ensure that the most appropriate model is chosen to address the research question(s). Researchers can control for injury severity and target damage to different brain regions by selection of appropriate models.

Careful study planning and thoughtful consideration are necessary to promote clinical relevance of animal studies of TBI. Specifically, attention should be given to selecting clinically relevant outcome variables that are comparable to what is observed in human patients. There are obvious challenges in assessing cognition in animals, compared to humans; for example, verbal assessments are not possible. Moreover, in deciding the types of long-term outcomes to evaluate in pre-clinical studies, researchers must have an understanding of the types of functional deficits that are commonly experienced by, and particularly distressing for, TBI survivors. Notably, some commonly reported problems, such as difficulties in devoting attention to multiple competing stimuli, cannot be easily studied in animals ³⁰⁶ ; however, Corina Bondi and her colleagues have proposed and tested one such assessment. ²⁴⁶

Assessment of functional outcomes in animal models requires inference in line with classic Skinnerian thought. As mentioned above, there are some functional outcomes of TBI in humans that have no clear analog in animals. Additional efforts are needed to map human to animal domains and develop additional reliable, valid assessments of functionality for use in animals. Moreover, it is worth noting that functional tests can be completed within hours in humans, whereas assessment of neurobehavioral function in animals can take much longer.

There are some relevant considerations for researchers regarding the species to use. Not all outcome variables can be assessed in all species. For example, if behavioral tests are of interest, a rodent model may be preferred given that functional outcome tests are well characterized in this context, especially when compared to large animals. ³⁰⁷ Rodent models afford unique opportunities to assess histopathological and behavioral consequences of TBI under controlled conditions and at precisely timed intervals. However, these small animal models are associated with their own set of pitfalls, such as limited generalizability, the potential for test strains to exhibit deficits that would confound study results, and the need for substantial research efforts before translation. ^308,309 Most behavioral tests developed for use in rats have been scaled for translation to mice; however, the tests may not yield similar injury effects across species.

Likewise, selection of assessment time point(s) can affect results. If assessed too early, the effects of anesthesia may confound findings, whereas, if the assessment occurs too late, the injury effect may have subsided. Thus, Fujimoto and colleagues suggest that researchers should assess the functional outcomes of interest at multiple time points. Previously, experimental TBI researchers have suggested that additional long-term studies are needed. ³¹⁰

Overall, experimental models have contributed substantially to our understanding of acute and chronic TBI outcomes. Despite available research, chronic changes remain relatively understudied, though available evidence suggests that chronic outcomes of experimental TBI are diverse and include histopathological and behavioral changes. In this review, effort was given to include as much evidence as possible; however, this review is not intended to be fully comprehensive. Readers of this review will take away a familiarization of key evidence from four common experimental TBI models (FPI, CCI, bTBI, and CHI), along with the key assessments used to evaluate the long-term histopathological and behavioral consequences of these models.