This study was undertaken to assess the efficacy of minocycline to treat mild repetitive head injury. Minocycline is reported to reduce microgliosis in mice and rats following significant brain damage caused by traumatic head impact [2, 18, 36, 43, 47]. All of these studies focused on the consequences of brain damage (e.g., lesion volume) and the subsequent loss of cognitive and motor behavior. The present study contributes to this body of literature in two ways. First, by using a model of mild repetitive head injury that better reflects the human experience. Head impacts were delivered during the dark period of the circadian cycle when rats are active, and then when rats were fully awake without the confound of anesthesia. There was no neuroradiological evidence of skull damage or brain contusion or noticeable deficits in motor behavior after each of three impacts. All of these findings attest to the mild nature of the head injury. Changes in diffusion weighted imaging, specifically increases in fractional anisotropy, were used as a surrogate measure of cytotoxic edema. These data are discussed below in the context of the many preclinical rodent studies on TBI with minocycline treatment and their translation to the human experience and clinical condition.
There have been numerous studies using minocycline to treat TBI in mice and rats [2, 5, 18, 36, 41, 43, 50, 55, 58]. All have used controlled cortical impact or weight drop protocols on anesthetized animals with open or closed skulls producing frank brain damage. The doses range from 20–90 mg/kg with various dosing regimens. Minocycline treatment under these conditions reduces neuroinflammation and microglia activation [2, 5, 17, 18, 41]. Alterations in emotion [5], motor [18] and olfactory function [50] are corrected with minocycline. Although, a study by Vonder Haar and coworkers using a dose regimen meant to mimic clinical practice reported only modest results with respect to restoration of behavioral functions [58]. Similarly, Pechacek et al. reported deficits in motor impulsivity and attention following head injury were unaffected by minocycline treatment [43], raising questions about the efficacy of minocycline for the treatment of psychiatric disorders following head injury.
The dosing regimen used by Taylor et al. in male and female adult Sprague Dawley rats was very similar to that used in the present study. Following an open skull CCI injury, rats were treated with 50 mg/kg of minocycline once daily for three consecutive days. The protracted hyperthermia caused by the TBI was reduced with minocycline treatment [55]. Kovesdi et al. used mild blast injury in anesthetized adult male Sprague Dawley rats maintained on a reverse L-D cycle and reported enhanced neuroinflammation and deficits in cognition and emotion [29]. Daily IP injections of 50 mg/kg of minocycline over four days reduced the biomarkers of inflammation and mitigated the behavioral deficits.
From all of these preclinical studies on TBI in rodents the ones most relevant to the findings in this study are those involved in measuring edema and the integrity of the blood brain barrier (BBB). Enhanced neuroinflammation, elevated proinflammatory cytokines and disruption in the BBB is common with most head injuries [11]. Homsi et al provided the first evidence that a specific treatment regimen of minocycline could reduce brain edema in mice following head injury [17]. More recently, Lu and coworkers reported a 45 mg/kg dose of minocycline given within 30 min of head injury reduced edema and preserved BBB integrity in mice. In another example using a different method other than head impact to cause brain injury, mice exposed to the neurotoxin 1,2-dicholorethane show many of the same pathological sequalae of head injury characterized by an increase in proinflammatory cytokines, gliosis, disruption in BBB integrity and edema. Treating mice with 45 mg/kg minocycline one hr before 1,2-DCE exposure reduces the edema [61].
Edema makes a significant contribution to the neuropathology of head injury [27, 56]. Vasogenic edema is caused by injury to the BBB and the immediate translocation of fluid to the extracellular space of the brain parenchyma. An increase in ADC, a quantitative measure of water mobility, is used as a surrogate marker for this change in extracellular volume [56]. The increase in ADC is usually accompanied by a decrease in FA. If the head injury is moderate or severe, cytogenic edema occurs characterized by cellular swelling due to loss of homeostatic regulation of osmolarity across the plasma membrane. This phase of brain edema usually presents with a decrease in ADC and increase in FA [23]. This increase in brain water contributes to parenchymal swelling and increase in intracranial pressure. Changes in BBB permeability and subsequent cerebral edema is dynamic with acute and chronic phases. For example, Logsdon and colleagues showed two mild blast injuries that cause an immediate increase in BBB over much of the brain, resolving with 24 h only to return 72 hrs later [35]. The vulnerable areas were the prefrontal cortex, hippocampus, thalamus, and medulla. In brain injury with contusion, Ren and Lu used DWI at multiple times over 72 hrs to follow the dynamic changes in edema in rats[45]. The initial vasogenic edema at 1 hr evolved into a combination of vasogenic and cytotoxic edema by 12 hrs that resolved by 24 hrs but reappeared after 48 that was prominently cytotoxic edema by 72 hrs.
In a previous study we reported a single mild impact devoid of any neuroradiological evidence of brain damage causes a short-lived increase in vasogenic edema in the thalamus, basal ganglia and cerebellum as evidenced by an increase in ADC [30]. The increase in extracellular fluid volume peaked at 6 hrs but returned to baseline by 24 hrs. In the present study female rats were subjected to three mild head impacts and imaged for changes in ADC and FA within a few hours of the last insult. We anticipated the severity of the vasogenic edema in these animals would be greater than that of a single impact characterized by an increase in ADC and decrease in FA. Indeed, the level of putative injury based on measures of DWI was widespread as shown in Fig. 2. To our surprise, the expected increase in ADC and decrease in FA was not realized; instead, all affected brain regions (e.g., thalamus, prefrontal ctx, hippocampus, cerebellum, basal ganglia and sensorimotor cortices) presented with little change in ADC but robust increases in FA. In all cases, treatment with minocycline reversed the increase in FA measures. As shown in Fig. 3, many of the same brain areas identified as sustaining a putative cytotoxic injury were treated with minocycline. Areas less responsive to minocycline were the midbrain dopaminergic system and the thalamus, raising questions about the sensitivity and vulnerability of these areas to head injury. Cai et all reported two mild head impacts in anesthetized male rats interrupted perivascular clearance and aquaporin 4 expression in the substantia nigra [3]. Interestingly, patients with mild-to-moderate TBI with persistent symptoms of diminished cognitive function present with higher FA values and lower ADC values in the midbrain[16].
Data Interpretation and Limitations
The major limitation in this study was the absence of males to assess sex differences in this model of repetitive mild head injury. In previous studies using anesthetized male Sprague Dawley rats we interrogated the brain injury with several different imaging modalities including a quantitative ultra short time to echo-contrast enhanced procedure to assess blood brain barrier disruption at the level of the microvasculature using the contrast agent ferumoxytol and resting state functional connectivity. This project was designed to evaluate head injury in awake animals with and without minocycline treatment using DWI alone, a modality we have run in all of our previous studies and an MRI procedure readily performed in the clinic. While the changes in DWI would suggest brain injury caused by edema, there were no significant changes in gliosis that would confirm the presence of neuroinflammation. As noted, the histological analysis was limited to 3–4 subjects and may have been underpowered.
Summary
A recent review by Cox et al. questioned the validity of preclinical models in guiding the development of new therapeutics for the treatment of head injury [10], a view shared by others to account for the many failed clinical trials for TBI [49, 51]. To that end we chose to eschew the standard models of TBI that routinely cause brain damage leading to measures of cognitive and motor dysfunction. Instead, we have focused on mild head injury common in organized sports, soldiers in combat and everyday accidents in the young and old. Critical to this model is the absence of any damage as confirmed by neuroradiology. The only evidence of injury is the “bump on the head” from the edema on the skin overlying the skull as shown in Fig. 1. This model, as reported in other studies on mild head injury [8, 25, 38, 46, 54], does not effectively alter behavior, discounting this measure as an endpoint when interpreting disease progression and drug efficacy using rodents. To make our model more relevant to the human experience, rats were head impacted while fully awake, eliminating the confound of anesthesia, and during the dark phase of their L-D cycle when they are most active. Magnetic resonance imaging for changes in indices of anisotropy using DWI and BBB permeability using blood contrast enhanced techniques are readily performed in the clinic, aiding in the translation of data from rats to humans by using the same techniques. Previous studies from our lab using imaging with mild impacts directed to the forehead have identified the thalamus, cerebellum, hippocampus, basal ganglia, and midbrain dopaminergic system as vulnerable areas [3, 30, 31, 33]. The data in this study is consistent with those findings, but using a model further refined to reflect the human experience of repetitive head injury. Moreover, we provide evidence using DWI that the alterations in gray matter microarchitecture affected by edema can be treated with minocycline given after head impact.