Testing the efficacy of minocycline treatment in an awake, female rat model of repetitive mild head injury

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As an antibiotic it reduces microglia activation. Can minocycline be used to treat mild head injury? To that end, minocycline was tested in a novel, closed-head, momentum exchange model of repetitive mild head injury in female rats impacted while fully awake. MRI revealed there was no brain damage or contusion attesting to the mild nature of the head impacts in this model. It was hypothesized that drug treatment would reduce edema and brain neuroinflammation. Female rats maintained on a reverse light-dark cycle were head impacted three times while fully awake with and without drug treatment. The impacts, separated by 24 hrs each, were delivered under red light illumination. Within 1-2 hrs of the last impact, rats were assessed for changes in water diffusion using diffusion weighted imaging. The data were registered to a 3D MRI rat atlas with 173 segmented brain areas providing site specific information on altered brain gray matter microarchitecture. Postmortem histology was performed 18 days post head injury. Head injury without minocycline treatment was characterized by multiple areas of increased fractional anisotropy, evidence of cytotoxic edema. Treatment with minocycline reversed these measures in many of the same areas and several others (e.g., hippocampus, basal ganglia, prefrontal cortex, sensory and motor cortices and thalamus). Histology for gliosis showed no evidence of neuroinflammation in the thalamus, hippocampus and cerebellum for control or experimental groups in this female model of mild head injury. These studies provide clear evidence that treatment with minocycline within hours after mild repetitive head injury significantly reduce measures of cytotoxic edema in a female rat model of mild repetitive head injury. concussion closed head momentum exchange microgliosis diffusion weighted imaging cytotoxic edema Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Minocycline is a tetracycline antibiotic commonly used to treat bacterial infections (e.g., acne), but has a potent anti-inflammatory effect independent of its antibacterial action [ 48 ] The anti-inflammatory effects of minocycline have been reported in various animal models of CNS disease like Alzheimer’s [ 7 ], Parkinson’s [ 12 ], Huntington’s [ 6 ] and traumatic brain injury [ 47 ]. There is compelling evidence that the anti-inflammation is caused by suppressing microglia activation; reducing inducible nitric oxide synthase (iNOS), an enzyme catalyzing the production of nitric oxide; and by decreasing matrix metalloproteases associated with blood brain barrier (BBB) permeability [ 52 ]. There is ever growing evidence that failure in the BBB lies at the foundation of cerebral small vessel disease (cSVD), the underlying pathophysiological process affecting arterioles, capillaries, and venules [ 42 , 60 ]. Cerebral SVD is a leading cause of dementia [ 21 , 24 , 59 ] and thought to be a significant source of neurological disability in aging and a key pathogenic factor in Alzheimer’s [ 9 ]. Indeed, there is an ongoing clinical trial testing the efficacy of minocycline to reduce inflammation and BBB leakage in small vessel disease (MINERVA) [ 1 ]. These conditions of neuroinflammation, microglia activation, and BBB leakage have been reproduced in a model of mild repetitive head injury using anesthetized male rats [ 31 ], (i.e., “the bump on the head” incurred while playing organized sports, car accidents, falls, or in military combat [ 20 , 32 , 34 ]). This closed head, momentum exchange model was developed to replicate the human experience of a mild head injury without neuroradiological evidence of damage to the skull or brain [ 30 ]. While the impacts are mild they result in changes in BBB permeability [ 33 ] and sustained neuroinflammation and microglia activation in heterogenous brain areas, specifically the midbrain dopaminergic system [ 3 ]. The present study was undertaken to advance the model to include female rats, impacted while fully awake and during the dark phase when they are active, and to evaluate the treatment effect of minocycline using magnetic resonance imaging (MRI). Given the effectiveness of minocycline in various rodent models of neurodegenerative diseases, we hypothesized it would have a beneficial effect in this model of mild head injury. Minocycline treatment selectively reduced measures of water diffusion that would suggest alteration in the gray matter microarchitecture associated with cytotoxic edema. Methods Animals Twenty-four Sprague Dawley female rats (250-300g) were purchased from Charles River Laboratories (Wilmington, MA, USA), housed on a reverse 12:12 light-dark cycle (lights off at 9:00 hr), maintained in ambient temperature (22–24°C) and provided with food and water ad libitum. All experiments were conducted under dim red illumination between 10:00 hrs and 18:00 hrs to avoid the transitions between the L-D dark cycles. Rats were randomly assigned to three experimental groups: 1) vehicle treated shams with no head injury (n = 8), 2) vehicle treated with head injury (n = 8), and 3) minocycline treated with head injury (n = 8). Minocycline hydrochloride (Thermo Fisher Scientific, Waltham. MA) was taken up in saline and given IP in a dose of 45 mg/kg in a volume of 1 mL/kg within 1 hr post head impact. The dose was taken from the literature using minocycline (45–50 mg/kg) to treat TBI in rats [ 29 , 43 , 55 ]. All animals were cared for in accordance with the NIH Guide to the Care and Use of Laboratory Animals. Methods and procedures used in this study were pre-approved by the Northeastern University Institutional Animal Care and Use Committee protocol 21-0824R. The protocols used in this study complied with the regulations of the institution and adhere to the ARRIVE guidelines for reporting in vivo experiments in animal research [ 28 ]. Mild Head Impact Head impacts were generated with a pneumatic pressure driven 50 g compactor described by Viano and colleagues [ 57 ] and refined by Mychasiuk et al [ 38 ] to reliably produce the 7.4 m/s impact velocities described for mild rat head injury. The kinetic energy at impact is 1.37 joules. We have used this model to publish on the long-term neuroradiological effects of repetitive mild head impacts in isoflurane anesthetized, male rats[ 3 , 31 ]. This model is comparable to CHIMERA developed for mouse mild head injury [ 39 , 40 ]. Before the first impact, all rats were treated with 0.1 mg/kg slow-releasing buprenorphine analgesic. The impact piston was directed to the top of the skull, midline, in the approximate area of Bregma while female rats were fully awake. All rats showed normal ambulatory behavior within seconds of being placed into their home cage after head impact. There were no mortalities. There was no evidence of skull damage or contusion (Fig. 1 ). Rats were subjected to three mild head impacts separated by 24 hour each as previously described [ 31 , 33 ]. All rats were imaged for edema using DWI within 1–2 hours of the third head impact. Eighteen days following the last impact rats were euthanized and the brain harvested for histology. Imaging Imaging sessions were conducted using a Bruker Biospec 7.0 T/20-cm USR horizontal magnet (Bruker, Billerica, MA, USA) and a 2 T/m magnetic field gradient insert (ID = 12 cm) capable of a 120-µs rise time. Radio frequency signals were sent and received with a quadrature volume coil built into the animal restrainer (Ekam Imaging, Boston MA, USA) [ 14 ]. The design of the restraining system included a padded head support obviating the need for ear bars, helping to reduce discomfort while minimizing motion artifact. All rats were imaged under 1–2% isoflurane while keeping a respiratory rate of 40–50 breaths/min. At the beginning of each imaging session, a high-resolution anatomical data set was collected for assessment of structural damage using the RARE pulse sequence with following parameters: 35 slice of 0.7mm thickness; field of view (FOV) 3 cm; 256×256; repetition time (TR) 3900 msec; effective echo time (TE) 48 msec; number of excitations (NEX) 3; 6 min 14 sec acquisition time. Diffusion Weighted Imaging – Quantitative Anisotropy DWI was acquired with a spin-echo echo-planar-imaging (EPI) pulse sequence having the following parameters: TR/TE = 500/20 msec, eight EPI segments, and 10 non-collinear gradient directions with a single b-value shell at 1000 s/mm 2 and one image with a B-value of 0 s/mm 2 (referred to as B0) as previously described [ 4 , 15 , 31 ]. Geometrical parameters were: 48 coronal slices, each 0.313 mm thick (brain volume) and with in-plane resolution of 0.313×0.313 mm 2 (matrix size 96×96; FOV 30 mm 3 ). The imaging protocol was repeated two times for signal averaging. Each DWI acquisition took 35 min and the entire MRI protocol including the anatomy lasted about 90 min. There are numerous studies detailing the benefits of multi-shot EPI in BOLD imaging [ 19 , 26 , 37 , 44 , 53 ]. We avoided using single shot EPI because of its severe geometrical distortion at high field strengths (≥ 7T) and loss of effective spatial resolution as the readout period increases [ 13 , 19 , 22 ]. There is also the possibility of signal loss in single shot EPI due to accumulated magnetic susceptibility or field inhomogeneity [ 26 ]. DWI analysis was completed with MATLAB and MedINRIA (1.9.0; http://www-sop.inria.fr/asclepios/software/MedINRIA/index.php ) software. Because sporadic excessive breathing during DWI acquisition can lead to significant image motion artifacts that are apparent only in the slices sampled when motion occurred, each image (for each slice and each gradient direction) was screened prior to DWI analysis. For statistical comparisons among rats, each brain volume was registered to the 3D MRI rat brain atlas allowing voxel- and region-based statistics. All image transformations and statistical analyses were carried out using the in-house EVA software (Ekam Solutions LLC, Boston MA). For each rat, the B0 image was co-registered with the MRI atlas using a 9-parameter affine transformation. Insight Toolkit ( https://itk.org/ ) registration framework was used with Affine transformation, mutual information similarity matrix and gradient descent optimizer with following parameters: Max step length 0.3 mm, Min step length 0.0001 mm, Number of iterations 100, Scan threshold 20%. Finally, before segmentation, registration of the scans were closely inspected for quality and manually corrected if necessary. The average value for each ROI was computed using map files for indices of apparent diffusion coefficient (ADC) and fractional anisotropy (FA). For statistical comparisons among rats, each brain volume was registered to a 3D MRI Rat Brain Atlas (© 2012 Ekam Solutions LLC, Boston, MA) allowing voxel- and region-based statistics. All image transformations and statistical analyses were carried out using the in-house MIVA software ( http://ccni.wpi.edu/ ). For each rat, the B0 image was co-registered with the B0 template (using a 6-parameter rigid-body transformation). The co-registration parameters were then applied on the DWI ADC and FA maps. Normalization was performed on the maps because they provided the most detailed visualization of brain structures and allowed for more accurate normalization. The normalization parameters were then smoothed with a 0.3-mm Gaussian kernel. To ensure that ADC and FA values were not affected significantly by the pre-processing steps, the ‘nearest neighbor’ option was used following registration and normalization. Statistical differences in measures of DWI between experimental groups were determined using one-way ANOVA followed by Bonferroni post hoc tests (alpha set at 5%). Statistical differences in measures of DWI between experimental groups were determined using mixed effects analyses followed by Tukey’s post hoc test. The 3D MRI rat atlas has 173 segmented, annotated brain areas. For DWI analysis (see below), 150 areas were chosen because they could be organized into well-defined neuroanatomical regions. Areas excluded were white matter tracts because they traverse several brain regions. Circumventricular organs (e.g., anterior and posterior pituitary, pineal gland, area postrema, median eminence) and small adjacent areas like the arcuate and retrochiasmatic nuclei, were also excluded because of their larger, more fenestrated blood vessels. Also excluded were areas with no clear regional organization (e.g., prerubral field). The remaining 150 brain areas were divided into 11 brain regions: cerebellum (20), cortex (19), thalamus (20), basal ganglia (10), hypothalamus (14), hippocampus (9), prefrontal cortex (9), olfactory bulb/cortex (8), amygdala (8), midbrain/pons (12), brainstem (21). The organization was based on conventional neuroanatomy and an effort to keep individual brain areas localized and contiguous within a region. The olfactory bulb/cortex is the exception as the piriform cortex extends some distance caudally along the ventral lateral cortex away from the bulbs. Immunohistochemistry Following perfusion and tissue collection, brains were sectioned and immunohistochemically stained for visualization of astrocyte (GFAP + ) and microglia (IBA1 + ) populations. Sections were obtained at 50 µm increments using a cryostat (Leica Biosystems) at -20°C and refrigerated at 4°C until staining. From each sectioned brain, 2–3 representative samples were selected per region of interest (thalamus, hippocampus, substantia nigra, and cerebellum) approximating the coordinates shown in Fig. 4 . Free-floating immunohistochemistry was conducted in 12-well plates on a lightly rotating shaker at 4°C. Sections were triple-rinsed in PBS (5 min/rinse) and blocked in 0.2% Triton X-100 (85111; ThermoFisher Scientific, Rockford, IL, USA) and 5% Normal Goat Serum (S26; EMD Millipore, Temecula, CA, USA) in PBS for 1 hour. After blocking, sections were incubated in primary antibody solution overnight for 18 hours [1:1000 rabbit anti-GFAP (Z0334; Agilent Dako, Cedar Creek, TX, USA) or 1:200 rabbit anti-AIF1/IBA1 (SAB5701363; Sigma-Aldrich, St. Louis, MO, USA) in blocking solution]. After primary antibody incubation, sections were triple-rinsed in PBS before incubation in secondary fluorescent antibody solution [1:400 goat anti-rabbit Alexa Fluor 488 (111-545-003; Jackson ImmunoResearch, West Grove, PA, USA) in blocking solution] for 1 hour. Following secondary antibody incubation, sections were triple-rinsed in PBS and mounted onto microscope slides in distilled water, cover slipped using Fluoroshield with DAPI mounting medium (F6057; Sigma-Aldrich, St. Louis, MO, USA), and sealed with clear polish. Slides were allowed to set for at least 24 hours at 4°C before confocal microscopy. Images were acquired using a Zeiss LSM 800 confocal microscope (Carl Zeiss Meditec AG, Jena, Germany; housed in the Institute for Chemical Imaging of Living Systems at Northeastern University) at 100X magnification. Data were collected from consistent sites within the thalamus, hippocampus, substantia nigra, and cerebellum (Fig. 4 ). GFAP or IBA1 signal intensity was measured using FIJI ImageJ at a consistent threshold. One-way ANOVA for each region was conducted using IBM SPSS Statistics and graphed using Prism GraphPad software. Results Diffusion Weighted Imaging Table 1a shows the significant changes in measures of FA between vehicle treated sham rats and rats that were untreated and hit. In this case the increase in FA is considered a surrogate measure of cytotoxic edema. This comparison shows the extent of head injury as 50/173 brain areas presented with an increase in FA values without treatment. These areas are ranked in order of their significance. Reported are the mean (highlighted in gray) and standard deviation (SD) together with their probability values and the omega square (ω Sq) for effect size. The critical value was set at p < 0.05 A false discovery rate (FDR) for multi-comparisons gave a significant level of p = 0.057. Table 1b lists 39/173 brains areas that showed a significant decrease in FA values with minocycline treatment. The FDR was p = 0.039. The location of many of these brains areas are shown in the 2D maps and summarized in the 3D reconstructions in Fig 3 . When sham untreated rats were compared with head injured rats treated to minocycline there were very few significant differences (See Supplementary File S5 ). Table 2a shows the significant changes in measures of ADC between vehicle treated shams and rats that were untreated and hit. In this case an increase in ADC would be interpreted as an increase in vasogenic edema. This comparison shows that only 7/173 brain areas presented with a change in ADC values. The critical value was set at p < 0.05. An FDR for multi-comparisons gave a significant level of p = 0.008. Table 2b lists 48/173 brains areas that showed a significant decrease in ADC values with minocycline treatment. All DWI measures for each experimental condition and their tables for all 173 brain areas are provided in Supplementary Data Files S1-S4 . Shown in Fig 2 are scatter plot/bar graphs (mean ± SD) for only brain regions that showed minocycline associated differences in FA and ADC. The dots represent each brain area contributing to that brain region. For example, the hippocampus is composed of nine areas: dorsal and ventral subiculum, dorsal and ventral dentate, dorsal and ventral CA3 and CA1 and a single CA2. For a list of brain areas in each region see Supplementary File S6 . With the data organized into brain regions there is a significant increase in ADC values associated with vasogenic edema when comparing hit with no treatment (CTL) to sham no hit rats (SHAM) for the thalamus (p<0.0001), cerebellum (p<0.001) and sensorimotor cortices (p<0.05). FA values were significantly greater in CTL than SHAM for all brain regions indicative of cytotoxic edema with repeated head injury. What was consistent across all groups, with the exception of the olfactory system, was a decrease in measures of FA and ADC with minocycline treatment (MINO) in head injured rats compared untreated head impacted controls, suggesting a decrease in vasogenic and cytotoxic edema. Fig 3 shows the anatomical localization of the brain areas listed in Tables 1a and b for FA values, presented as 2D activation maps. The coronal sections are labeled (a.) through (h.) and arranged from rostral (top) to caudal (bottom). The red denotes brain areas where FA values were significantly increased with head injury but no treatment. The blue denotes brain areas where FA values were significantly decreased in head injured rats treated with minocycline. Areas in yellow denote the location of white matter tracts. In brain section (a.), the olfactory bulb with three different layers shows injury to the glomerular and granular layers. Treatment with minocycline significantly reduces FA values or the putative cytotoxic edema in the glomerular layer shown in blue. Brain section (b.) highlights the injury in the forebrain prefrontal ctx (e.g., prelimbic, infralimbic and ventral orbital cortices) shown in red. Treatment with minocycline reversed the FA values in all of these injured areas with the exception of the prelimbic ctx and somatosensory ctx. Brain section (c.) highlights injury to the dopaminergic, forebrain basal ganglia (e.g., dorsal lateral striatum, medal and lateral ventral striatum, accumbens core and shell, and ventral pallidum) shown in red. Treatment with minocycline reversed FA values in all of these injured sites with the exception of somatosensory ctx and ventral pallidum. Indeed, as you progress through each of the brain sections the areas of putative cytotoxic injury defined by an increase in FA shown in red also appear as blue with minocycline treatment, but not all injured areas are recovered with treatment. This distinction is shown in the 3D reconstructions to the right. The red reconstruction shows the injured whole brain (i.e., significantly elevated levels of FA suggestive of cytotoxic edema) and the blue reconstruction represents injured areas sensitive to minocycline treatment (i.e., significantly reduced measures of FA). The reconstructions are not identical. It should be noted that areas in brainstem sections (g. & h.) show significant decreases in FA values (e.g., sensory n. trigeminal, central gray, pontine reticular n. and 9 th cerebellar lobule) (blue) that are not identified as having putative cytotoxic edema (no red). However, there are several areas of the hit, untreated brain that are not “recovered” with minocycline treatment (i.e., red with no matching blue). These areas are shown in the boxed insert. Of note are the thalamus and the substantia nigra and VTA. Immunohistochemistry Shown in Fig 4 are representative micrographs of immunostaining for gliosis from the three experimental groups. No significant main effects of experimental condition were observed in any region ( Table 3 ). A post hoc power analysis conducted using G*Power software indicated an achieved power of 1-b = 0.153 to detect a large effect, an important limitation of this analysis. Discussion 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. Declarations Conflict of Interest : The CFF has a financial interest in Animal Imaging Research, a company that makes the radiofrequency electronics and holders for awake animal imaging. The CFF and PK have a partnership interest in Ekam Solutions the company that develops 3D MRI atlases for animal research. Authors' Contributions: All of the authors have contributed substantially to the manuscript. All authors have read and agreed to the published version of the manuscript. Concept, drafting and interpretation – Hightower, Ferris, Kulkarni, Execution and analysis – Hightower, Prom, Brengel. Funding: This work was supported by Elam Imaging, Inc. Acknowledgements: We owe a dept of gratitude to Dr. Andrew Schafer whose scholarship supported the training and research of Ms. Rosemarie Hightower, an undergraduate student enrolled in the College of Science, Northeastern University. Data Availability : All data available on request References Brown R, Tozer D, Loubiere L, Hong Y, Fryer T, Williams G, Graves M, Aigbirhio F, O'Brien J, Markus H (2022) MINocyclinE to Reduce inflammation and blood brain barrier leakage in small Vessel diseAse (MINERVA) trial study protocol. European Stroke Journal: 1-8 Doi DOI: 10.1177/23969873221100338 Bye N, Habgood MD, Callaway JK, Malakooti N, Potter A, Kossmann T, Morganti-Kossmann MC (2007) Transient neuroprotection by minocycline following traumatic brain injury is associated with attenuated microglial activation but no changes in cell apoptosis or neutrophil infiltration. 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Cells 10: Doi 10.3390/cells101026 Tables Table 3 is not available with this version Tables 1 to 2 are available in the Supplementary Files section Supplementary Files Supplementary Files S5-S6 are not available with this version Supplementary Files SupplementaryFilesS1FAVehShamvsVehHitTable.xlsx SupplementaryFilesS2FAVehhitvsMinoHit.xlsx SupplementaryfilesS3ADCShamvehvsHitVeh.xlsx SupplementaryFilesS4ADCVehvsMinoHit.xlsx Tables.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 17 Apr, 2024 Reviewers invited by journal 17 Apr, 2024 Editor assigned by journal 09 Apr, 2024 First submitted to journal 08 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4228869","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":292158734,"identity":"e7fdc929-48bf-4260-b99a-e330877255a2","order_by":0,"name":"Rosemarie Hightower","email":"","orcid":"","institution":"Northeastern University - Boston Campus: Northeastern University","correspondingAuthor":false,"prefix":"","firstName":"Rosemarie","middleName":"","lastName":"Hightower","suffix":""},{"id":292158735,"identity":"84b6163d-5917-4da9-8a3d-6a32871be569","order_by":1,"name":"Eric Brengel","email":"","orcid":"","institution":"Northeastern University - Boston Campus: Northeastern University","correspondingAuthor":false,"prefix":"","firstName":"Eric","middleName":"","lastName":"Brengel","suffix":""},{"id":292158736,"identity":"757d7512-13e8-4864-aa71-9c77607ea2d6","order_by":2,"name":"Sophia Prom","email":"","orcid":"","institution":"Northeastern University - Boston Campus: Northeastern University","correspondingAuthor":false,"prefix":"","firstName":"Sophia","middleName":"","lastName":"Prom","suffix":""},{"id":292158737,"identity":"3a38ef2a-bacb-439a-b02a-1c9b69c72ff6","order_by":3,"name":"Praveen Kulkarni","email":"","orcid":"","institution":"Northeastern University - Boston Campus: Northeastern University","correspondingAuthor":false,"prefix":"","firstName":"Praveen","middleName":"","lastName":"Kulkarni","suffix":""},{"id":292158738,"identity":"85f41879-119d-48d7-96a3-b3236252003b","order_by":4,"name":"Craig Ferris","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIie3OIWsDMRTA8Rce3MxBbCpKv8KVwJjoel9lR+DUxORqRuDg6qZTKu4rRI2JiRyBqM4XNjEozJeqqS4XNdGMwsxE/uIRwvvBA0il/mXkww8GNLzlj395ajuERSCjYaMfRnYeASjMuYRKJLvFy1XJ3xp32D/PJpPOP+5gNtbmNGEGcbr5ZNXTuxOq39RT7TKxVlDzGCmAupE07OZye8uhby3RWc4xB1vFCV58eVJyFcix7Fp68OT4G8mIJ0SzQEwlXY6emChhFnE4rFLbWsBrK4R2tT+sEHwVIXTZkL00DyVVwsKinV93jd1hfj8fP0YIYOzgVCqVSv2hb4MGWgmI7IZLAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-9744-5214","institution":"Northeastern University - Boston Campus: Northeastern University","correspondingAuthor":true,"prefix":"","firstName":"Craig","middleName":"","lastName":"Ferris","suffix":""}],"badges":[],"createdAt":"2024-04-06 23:01:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4228869/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4228869/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55174908,"identity":"231a1e0b-dce3-4229-900c-bc133856c7db","added_by":"auto","created_at":"2024-04-23 16:07:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":234663,"visible":true,"origin":"","legend":"\u003cp\u003eNeuroradiology\u003c/p\u003e\n\u003cp\u003eShown are radiograms of each subject in each experimental group. The edema (yellow arrow) that appears on the skin and tissue overlying the skull (dark area noted by white arrow) appears white using a T2 weighted pulse sequence. One rat in the no treatment impact group did not present with edema at the putative site of impact (see asterisk). In all other cases the impact site and edema at the forebrain (level of Bregma) was consistent across all rats following the 3rd impact except for the last rat in the minocycline treated group where the impact sited is more rostral near the cerebrum (see asterisk). While all sham rats had no hits, one showed some evidence of edema that could not be accounted for (see asterisk).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4228869/v1/bf0cad47aa64658c23d39af8.png"},{"id":55174910,"identity":"b863a5e4-dcfe-48f3-8afb-7d8dda2f2bbf","added_by":"auto","created_at":"2024-04-23 16:07:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":516517,"visible":true,"origin":"","legend":"\u003cp\u003eRegional changes in water diffusivity using DWI\u003c/p\u003e\n\u003cp\u003eShown are bar graphs (mean ± SD) and dot plots for different brain regions for two indices of anisotropy, apparent diffusion coefficient (ADC) and fractional anisotropy (FA), for each experimental condition. \u0026nbsp;The dots represent the number of brain areas in that particular brain region. For example, the hippocampus, comprised of the dorsal and ventral dentate, dorsal and ventral subiculum, dorsal and ventral CA1, dorsal and ventral CA3, and CA2, has nine dots. Each dot is the average of the values for that particular brain area in shams without head injury (n=8), rats hit three times with no treatment (n=7) and rats hit three times and treated with minocycline (n=8). The list of brain areas that comprise prefrontal ctx, basal ganglia, cerebellum, thalamus, and sensory and motor cortices can be found in \u003cstrong\u003eSupplementary Data S1\u003c/strong\u003e: *\u0026lt;0.05; ***\u0026lt;0.001***; ****\u0026lt;0.0001\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4228869/v1/c8fcbfcfaad59d31f23e8908.png"},{"id":55174909,"identity":"2b845f66-5553-4afe-bc4c-888807ea7ee0","added_by":"auto","created_at":"2024-04-23 16:07:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2213101,"visible":true,"origin":"","legend":"\u003cp\u003eFractional Anisotropy Heat Maps\u003c/p\u003e\n\u003cp\u003eShown are the anatomical localization of the brain areas listed in \u003cstrong\u003eTables 1a and b \u003c/strong\u003efor FA values, presented as 2D activation maps. The coronal sections are labeled (a.) through (h.) and arranged from rostral (top) to caudal (bottom). The red denotes brain areas where FA values were significantly increased with head injury but no treatment. The blue denotes brain areas where FA values were significantly decreased in head injured rats treated with minocycline. Areas in yellow denote the location of white matter tracts. There are several areas of the hit, untreated brain that are not represented in the minocycline treated maps (i.e., red with no matching blue). These areas are shown in the boxed insert.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4228869/v1/be84de8e04cde162275f131c.png"},{"id":55174915,"identity":"2a19cdcd-a0fc-42a3-a357-465397152ded","added_by":"auto","created_at":"2024-04-23 16:07:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1969170,"visible":true,"origin":"","legend":"\u003cp\u003eHistology\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea)\u003c/strong\u003e Data collection sites within the thalamus, hippocampus, and cerebellum are outlined in red, with the substantia nigra outlined in blue. \u003cstrong\u003eb)\u003c/strong\u003e \u0026amp; \u003cstrong\u003ec)\u003c/strong\u003e Signal intensity values are graphed for GFAP\u003csup\u003e+\u003c/sup\u003e astrocytes (b) and IBA1\u003csup\u003e+\u003c/sup\u003e microglia (c) in each region above corresponding sample images. Individual data points represent the average signal intensity of the region per subject, shaded bars signify each group mean, and error bars indicate the range of ±1 SD from the group mean.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4228869/v1/a7bc72258a74ab9c7824ec64.png"},{"id":55176671,"identity":"d38431b0-ff2e-4870-b404-2188a435e44e","added_by":"auto","created_at":"2024-04-23 16:24:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3559391,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4228869/v1/4cf04720-3711-4749-b2aa-a79b63c5f530.pdf"},{"id":55174912,"identity":"36b4f1d9-cf11-4c29-bcfd-7534c06ab3ab","added_by":"auto","created_at":"2024-04-23 16:07:59","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":59913,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFilesS1FAVehShamvsVehHitTable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4228869/v1/93d3725515c4be9a2f70f064.xlsx"},{"id":55174911,"identity":"9925a393-e161-4aa4-aa6d-af7c9697b3ca","added_by":"auto","created_at":"2024-04-23 16:07:58","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":70752,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFilesS2FAVehhitvsMinoHit.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4228869/v1/86f10b23831628d023cdc55a.xlsx"},{"id":55174916,"identity":"a7c29b81-8290-4501-8e8c-d0df2306ebde","added_by":"auto","created_at":"2024-04-23 16:07:59","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":55799,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryfilesS3ADCShamvehvsHitVeh.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4228869/v1/d0281a5c885244b3a573312c.xlsx"},{"id":55174917,"identity":"9a25c397-7d96-47cb-ab73-e91b38bcd0ff","added_by":"auto","created_at":"2024-04-23 16:07:59","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":75370,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFilesS4ADCVehvsMinoHit.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4228869/v1/4ddf51f6664133e3bb8a08cd.xlsx"},{"id":55175888,"identity":"fed03052-d517-4774-b19c-61d3c0c7250c","added_by":"auto","created_at":"2024-04-23 16:15:59","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":39635,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-4228869/v1/233c6a64b4e22dfbc3ec55af.docx"}],"financialInterests":"","formattedTitle":"Testing the efficacy of minocycline treatment in an awake, female rat model of repetitive mild head injury","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMinocycline is a tetracycline antibiotic commonly used to treat bacterial infections (e.g., acne), but has a potent anti-inflammatory effect independent of its antibacterial action [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] The anti-inflammatory effects of minocycline have been reported in various animal models of CNS disease like Alzheimer\u0026rsquo;s [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], Parkinson\u0026rsquo;s [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], Huntington\u0026rsquo;s [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and traumatic brain injury [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. There is compelling evidence that the anti-inflammation is caused by suppressing microglia activation; reducing inducible nitric oxide synthase (iNOS), an enzyme catalyzing the production of nitric oxide; and by decreasing matrix metalloproteases associated with blood brain barrier (BBB) permeability [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. There is ever growing evidence that failure in the BBB lies at the foundation of cerebral small vessel disease (cSVD), the underlying pathophysiological process affecting arterioles, capillaries, and venules [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Cerebral SVD is a leading cause of dementia [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] and thought to be a significant source of neurological disability in aging and a key pathogenic factor in Alzheimer\u0026rsquo;s [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Indeed, there is an ongoing clinical trial testing the efficacy of minocycline to reduce inflammation and BBB leakage in small vessel disease (MINERVA) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThese conditions of neuroinflammation, microglia activation, and BBB leakage have been reproduced in a model of mild repetitive head injury using anesthetized male rats [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], (i.e., \u0026ldquo;the bump on the head\u0026rdquo; incurred while playing organized sports, car accidents, falls, or in military combat [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]). This closed head, momentum exchange model was developed to replicate the human experience of a mild head injury without neuroradiological evidence of damage to the skull or brain [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. While the impacts are mild they result in changes in BBB permeability [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and sustained neuroinflammation and microglia activation in heterogenous brain areas, specifically the midbrain dopaminergic system [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The present study was undertaken to advance the model to include female rats, impacted while fully awake and during the dark phase when they are active, and to evaluate the treatment effect of minocycline using magnetic resonance imaging (MRI). Given the effectiveness of minocycline in various rodent models of neurodegenerative diseases, we hypothesized it would have a beneficial effect in this model of mild head injury. Minocycline treatment selectively reduced measures of water diffusion that would suggest alteration in the gray matter microarchitecture associated with cytotoxic edema.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eTwenty-four Sprague Dawley female rats (250-300g) were purchased from Charles River Laboratories (Wilmington, MA, USA), housed on a reverse 12:12 light-dark cycle (lights off at 9:00 hr), maintained in ambient temperature (22\u0026ndash;24\u0026deg;C) and provided with food and water ad libitum. All experiments were conducted under dim red illumination between 10:00 hrs and 18:00 hrs to avoid the transitions between the L-D dark cycles. Rats were randomly assigned to three experimental groups: 1) vehicle treated shams with no head injury (n\u0026thinsp;=\u0026thinsp;8), 2) vehicle treated with head injury (n\u0026thinsp;=\u0026thinsp;8), and 3) minocycline treated with head injury (n\u0026thinsp;=\u0026thinsp;8). Minocycline hydrochloride (Thermo Fisher Scientific, Waltham. MA) was taken up in saline and given IP in a dose of 45 mg/kg in a volume of 1 mL/kg within 1 hr post head impact. The dose was taken from the literature using minocycline (45\u0026ndash;50 mg/kg) to treat TBI in rats [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. All animals were cared for in accordance with the NIH Guide to the Care and Use of Laboratory Animals. Methods and procedures used in this study were pre-approved by the Northeastern University Institutional Animal Care and Use Committee protocol 21-0824R. The protocols used in this study complied with the regulations of the institution and adhere to the ARRIVE guidelines for reporting \u003cem\u003ein vivo\u003c/em\u003e experiments in animal research [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMild Head Impact\u003c/h2\u003e \u003cp\u003eHead impacts were generated with a pneumatic pressure driven 50 g compactor described by Viano and colleagues [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] and refined by Mychasiuk et al [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] to reliably produce the 7.4 m/s impact velocities described for mild rat head injury. The kinetic energy at impact is 1.37 joules. We have used this model to publish on the long-term neuroradiological effects of repetitive mild head impacts in isoflurane anesthetized, male rats[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. This model is comparable to CHIMERA developed for mouse mild head injury [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Before the first impact, all rats were treated with 0.1 mg/kg slow-releasing buprenorphine analgesic. The impact piston was directed to the top of the skull, midline, in the approximate area of Bregma while female rats were fully awake. All rats showed normal ambulatory behavior within seconds of being placed into their home cage after head impact. There were no mortalities. There was no evidence of skull damage or contusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Rats were subjected to three mild head impacts separated by 24 hour each as previously described [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. All rats were imaged for edema using DWI within 1\u0026ndash;2 hours of the third head impact. Eighteen days following the last impact rats were euthanized and the brain harvested for histology.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eImaging\u003c/h2\u003e \u003cp\u003eImaging sessions were conducted using a Bruker Biospec 7.0 T/20-cm USR horizontal magnet (Bruker, Billerica, MA, USA) and a 2 T/m magnetic field gradient insert (ID\u0026thinsp;=\u0026thinsp;12 cm) capable of a 120-\u0026micro;s rise time. Radio frequency signals were sent and received with a quadrature volume coil built into the animal restrainer (Ekam Imaging, Boston MA, USA) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The design of the restraining system included a padded head support obviating the need for ear bars, helping to reduce discomfort while minimizing motion artifact. All rats were imaged under 1\u0026ndash;2% isoflurane while keeping a respiratory rate of 40\u0026ndash;50 breaths/min. At the beginning of each imaging session, a high-resolution anatomical data set was collected for assessment of structural damage using the RARE pulse sequence with following parameters: 35 slice of 0.7mm thickness; field of view (FOV) 3 cm; 256\u0026times;256; repetition time (TR) 3900 msec; effective echo time (TE) 48 msec; number of excitations (NEX) 3; 6 min 14 sec acquisition time.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDiffusion Weighted Imaging \u0026ndash; Quantitative Anisotropy\u003c/h2\u003e \u003cp\u003eDWI was acquired with a spin-echo echo-planar-imaging (EPI) pulse sequence having the following parameters: TR/TE\u0026thinsp;=\u0026thinsp;500/20 msec, eight EPI segments, and 10 non-collinear gradient directions with a single b-value shell at 1000 s/mm\u003csup\u003e2\u003c/sup\u003e and one image with a B-value of 0 s/mm\u003csup\u003e2\u003c/sup\u003e (referred to as B0) as previously described [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Geometrical parameters were: 48 coronal slices, each 0.313 mm thick (brain volume) and with in-plane resolution of 0.313\u0026times;0.313 mm\u003csup\u003e2\u003c/sup\u003e (matrix size 96\u0026times;96; FOV 30 mm\u003csup\u003e3\u003c/sup\u003e). The imaging protocol was repeated two times for signal averaging. Each DWI acquisition took 35 min and the entire MRI protocol including the anatomy lasted about 90 min. There are numerous studies detailing the benefits of multi-shot EPI in BOLD imaging [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. We avoided using single shot EPI because of its severe geometrical distortion at high field strengths (\u0026ge;\u0026thinsp;7T) and loss of effective spatial resolution as the readout period increases [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. There is also the possibility of signal loss in single shot EPI due to accumulated magnetic susceptibility or field inhomogeneity [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDWI analysis was completed with MATLAB and MedINRIA (1.9.0; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www-sop.inria.fr/asclepios/software/MedINRIA/index.php\u003c/span\u003e\u003cspan address=\"http://www-sop.inria.fr/asclepios/software/MedINRIA/index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) software. Because sporadic excessive breathing during DWI acquisition can lead to significant image motion artifacts that are apparent only in the slices sampled when motion occurred, each image (for each slice and each gradient direction) was screened prior to DWI analysis. For statistical comparisons among rats, each brain volume was registered to the 3D MRI rat brain atlas allowing voxel- and region-based statistics. All image transformations and statistical analyses were carried out using the in-house EVA software (Ekam Solutions LLC, Boston MA). For each rat, the B0 image was co-registered with the MRI atlas using a 9-parameter affine transformation. Insight Toolkit (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itk.org/\u003c/span\u003e\u003cspan address=\"https://itk.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) registration framework was used with Affine transformation, mutual information similarity matrix and gradient descent optimizer with following parameters: Max step length 0.3 mm, Min step length 0.0001 mm, Number of iterations 100, Scan threshold 20%. Finally, before segmentation, registration of the scans were closely inspected for quality and manually corrected if necessary. The average value for each ROI was computed using map files for indices of apparent diffusion coefficient (ADC) and fractional anisotropy (FA).\u003c/p\u003e \u003cp\u003eFor statistical comparisons among rats, each brain volume was registered to a 3D MRI Rat Brain Atlas (\u0026copy; 2012 Ekam Solutions LLC, Boston, MA) allowing voxel- and region-based statistics. All image transformations and statistical analyses were carried out using the in-house MIVA software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://ccni.wpi.edu/\u003c/span\u003e\u003cspan address=\"http://ccni.wpi.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). For each rat, the B0 image was co-registered with the B0 template (using a 6-parameter rigid-body transformation). The co-registration parameters were then applied on the DWI ADC and FA maps. Normalization was performed on the maps because they provided the most detailed visualization of brain structures and allowed for more accurate normalization. The normalization parameters were then smoothed with a 0.3-mm Gaussian kernel. To ensure that ADC and FA values were not affected significantly by the pre-processing steps, the \u0026lsquo;nearest neighbor\u0026rsquo; option was used following registration and normalization. Statistical differences in measures of DWI between experimental groups were determined using one-way ANOVA followed by Bonferroni post hoc tests (alpha set at 5%). Statistical differences in measures of DWI between experimental groups were determined using mixed effects analyses followed by Tukey\u0026rsquo;s post hoc test.\u003c/p\u003e \u003cp\u003eThe 3D MRI rat atlas has 173 segmented, annotated brain areas. For DWI analysis (see below), 150 areas were chosen because they could be organized into well-defined neuroanatomical regions. Areas excluded were white matter tracts because they traverse several brain regions. Circumventricular organs (e.g., anterior and posterior pituitary, pineal gland, area postrema, median eminence) and small adjacent areas like the arcuate and retrochiasmatic nuclei, were also excluded because of their larger, more fenestrated blood vessels. Also excluded were areas with no clear regional organization (e.g., prerubral field). The remaining 150 brain areas were divided into 11 brain regions: cerebellum (20), cortex (19), thalamus (20), basal ganglia (10), hypothalamus (14), hippocampus (9), prefrontal cortex (9), olfactory bulb/cortex (8), amygdala (8), midbrain/pons (12), brainstem (21). The organization was based on conventional neuroanatomy and an effort to keep individual brain areas localized and contiguous within a region. The olfactory bulb/cortex is the exception as the piriform cortex extends some distance caudally along the ventral lateral cortex away from the bulbs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eFollowing perfusion and tissue collection, brains were sectioned and immunohistochemically stained for visualization of astrocyte (GFAP\u003csup\u003e+\u003c/sup\u003e) and microglia (IBA1\u003csup\u003e+\u003c/sup\u003e) populations. Sections were obtained at 50 \u0026micro;m increments using a cryostat (Leica Biosystems) at -20\u0026deg;C and refrigerated at 4\u0026deg;C until staining. From each sectioned brain, 2\u0026ndash;3 representative samples were selected per region of interest (thalamus, hippocampus, substantia nigra, and cerebellum) approximating the coordinates shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Free-floating immunohistochemistry was conducted in 12-well plates on a lightly rotating shaker at 4\u0026deg;C. Sections were triple-rinsed in PBS (5 min/rinse) and blocked in 0.2% Triton X-100 (85111; ThermoFisher Scientific, Rockford, IL, USA) and 5% Normal Goat Serum (S26; EMD Millipore, Temecula, CA, USA) in PBS for 1 hour. After blocking, sections were incubated in primary antibody solution overnight for 18 hours [1:1000 rabbit anti-GFAP (Z0334; Agilent Dako, Cedar Creek, TX, USA) or 1:200 rabbit anti-AIF1/IBA1 (SAB5701363; Sigma-Aldrich, St. Louis, MO, USA) in blocking solution]. After primary antibody incubation, sections were triple-rinsed in PBS before incubation in secondary fluorescent antibody solution [1:400 goat anti-rabbit Alexa Fluor 488 (111-545-003; Jackson ImmunoResearch, West Grove, PA, USA) in blocking solution] for 1 hour. Following secondary antibody incubation, sections were triple-rinsed in PBS and mounted onto microscope slides in distilled water, cover slipped using Fluoroshield with DAPI mounting medium (F6057; Sigma-Aldrich, St. Louis, MO, USA), and sealed with clear polish. Slides were allowed to set for at least 24 hours at 4\u0026deg;C before confocal microscopy.\u003c/p\u003e \u003cp\u003eImages were acquired using a Zeiss LSM 800 confocal microscope (Carl Zeiss Meditec AG, Jena, Germany; housed in the Institute for Chemical Imaging of Living Systems at Northeastern University) at 100X magnification. Data were collected from consistent sites within the thalamus, hippocampus, substantia nigra, and cerebellum (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003e). GFAP or IBA1 signal intensity was measured using FIJI ImageJ at a consistent threshold. One-way ANOVA for each region was conducted using IBM SPSS Statistics and graphed using Prism GraphPad software.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eDiffusion Weighted Imaging \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1a\u003c/strong\u003e shows the significant changes in measures of FA between vehicle treated sham rats and rats that were untreated and hit. \u0026nbsp;In this case the \u003cu\u003eincrease\u003c/u\u003e in FA is considered a surrogate measure of cytotoxic edema. This comparison shows the extent of head injury as 50/173 brain areas presented with an increase in FA values without treatment. These areas are ranked in order of their significance. Reported are the mean (highlighted in gray) and standard deviation (SD) together with their probability values and the omega square (ω Sq) for effect size. The critical value was set at p \u0026lt; 0.05 \u0026nbsp;A false discovery rate (FDR) for multi-comparisons gave a significant level of p = 0.057. \u0026nbsp; \u003cstrong\u003eTable 1b\u003c/strong\u003e lists 39/173 brains areas that showed a significant \u003cu\u003edecrease\u003c/u\u003e in FA values with minocycline treatment. \u0026nbsp;The FDR was p = 0.039. \u0026nbsp;The location of many of these brains areas are shown in the 2D maps and summarized in the 3D reconstructions in \u003cstrong\u003eFig 3\u003c/strong\u003e. When sham untreated rats were compared with head injured rats treated to minocycline there were very few significant differences (See \u003cstrong\u003eSupplementary File S5\u003c/strong\u003e). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2a\u003c/strong\u003e shows the significant changes in measures of ADC between vehicle treated shams and rats that were untreated and hit. \u0026nbsp;In this case an \u003cu\u003eincrease\u003c/u\u003e in ADC would be \u0026nbsp;interpreted as an increase in vasogenic edema. This comparison shows that only 7/173 brain areas presented with a change in ADC values. The critical value was set at p \u0026lt; 0.05. \u0026nbsp;An FDR for multi-comparisons gave a significant level of p = 0.008. \u0026nbsp; \u003cstrong\u003eTable 2b\u003c/strong\u003e lists 48/173 brains areas that showed a significant \u003cu\u003edecrease\u003c/u\u003e in ADC values with minocycline treatment. \u0026nbsp;All DWI measures for each experimental condition and their tables for all 173 brain areas are provided in \u003cstrong\u003eSupplementary Data Files S1-S4\u003c/strong\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eShown in \u003cstrong\u003eFig 2\u003c/strong\u003e are scatter plot/bar graphs (mean ± SD) for only brain regions that showed minocycline associated differences in FA and ADC. The dots represent each brain area contributing to that brain region. For example, the hippocampus is composed of nine areas: dorsal and ventral subiculum, dorsal and ventral dentate, dorsal and ventral CA3 and CA1 and a single CA2. For a list of brain areas in each region see\u0026nbsp;\u003cstrong\u003eSupplementary File S6\u003c/strong\u003e. \u0026nbsp;With the data organized into brain regions there is a significant increase in ADC values associated with vasogenic edema when comparing hit with no treatment (CTL) to sham no hit rats (SHAM) for the thalamus (p\u0026lt;0.0001), cerebellum (p\u0026lt;0.001) and sensorimotor cortices (p\u0026lt;0.05). FA values were significantly greater in CTL than SHAM for all brain regions indicative of cytotoxic edema with repeated head injury. \u0026nbsp; What was consistent across all groups, with the exception of the olfactory system, was a decrease in measures of FA and ADC with minocycline treatment (MINO) in head injured rats compared untreated head impacted controls, suggesting a decrease in vasogenic and cytotoxic edema.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003cstrong\u003eFig 3\u0026nbsp;\u003c/strong\u003eshows the anatomical localization of the brain areas listed in \u003cstrong\u003eTables 1a and b\u0026nbsp;\u003c/strong\u003efor FA values, presented as 2D activation maps. The coronal sections are labeled (a.) through (h.) and arranged from rostral (top) to caudal (bottom). The red denotes brain areas where FA values were significantly \u003cu\u003eincreased\u003c/u\u003e with head injury but no treatment. The blue denotes brain areas where FA values were significantly \u003cu\u003edecreased\u003c/u\u003e in head injured rats treated with minocycline. \u0026nbsp;Areas in yellow denote the location of white matter tracts. \u0026nbsp;In brain section (a.), the olfactory bulb with three different layers shows injury to the glomerular and granular layers. \u0026nbsp;Treatment with minocycline significantly reduces FA values or the putative cytotoxic edema in the glomerular layer shown in blue. Brain section (b.) highlights the injury in the forebrain prefrontal ctx (e.g., prelimbic, infralimbic and ventral orbital cortices) shown in red. Treatment with minocycline reversed the FA values in all of these injured areas with the exception of the prelimbic ctx and somatosensory ctx. \u0026nbsp;Brain section (c.) highlights injury to the dopaminergic, forebrain basal ganglia (e.g., dorsal lateral striatum, medal and lateral ventral striatum, accumbens core and shell, and ventral pallidum) shown in red. Treatment with minocycline reversed FA values in all of these injured sites with the exception of somatosensory ctx and ventral pallidum. \u0026nbsp;Indeed, as you progress through each of the brain sections the areas of putative cytotoxic injury defined by an increase in FA shown in red also appear as blue with minocycline treatment, but not all injured areas are recovered with treatment. \u0026nbsp;This distinction is shown in the 3D reconstructions to the right. The red reconstruction shows the injured whole brain (i.e., significantly elevated levels of FA suggestive of cytotoxic edema) and the blue reconstruction represents injured areas sensitive to minocycline treatment (i.e., significantly reduced measures of FA). \u0026nbsp;The reconstructions are not identical. \u0026nbsp;It should be noted that areas in brainstem sections (g. \u0026amp; h.) show significant decreases in FA values (e.g., sensory n. trigeminal, central gray, pontine reticular n. and 9\u003csup\u003eth\u003c/sup\u003e cerebellar lobule) (blue) that are not identified as having putative cytotoxic edema (no red). However, there are several areas of the hit, untreated brain that are not “recovered” with minocycline treatment (i.e., red with no matching blue). These areas are shown in the boxed insert. \u0026nbsp; Of note are the thalamus and the substantia nigra and VTA. \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cem\u003eImmunohistochemistry\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eShown in \u003cstrong\u003eFig 4\u003c/strong\u003e are representative micrographs of immunostaining for gliosis from the three experimental groups. \u0026nbsp; No significant main effects of experimental condition were observed in any region (\u003cstrong\u003eTable 3\u003c/strong\u003e). A \u003cem\u003epost hoc\u003c/em\u003e power analysis conducted using G*Power software indicated an achieved power of 1-b = 0.153 to detect a large effect, an important limitation of this analysis.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis 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 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. 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.\u003c/p\u003e \u003cp\u003eThere have been numerous studies using minocycline to treat TBI in mice and rats [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. 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\u0026ndash;90 mg/kg with various dosing regimens. Minocycline treatment under these conditions reduces neuroinflammation and microglia activation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Alterations in emotion [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], motor [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and olfactory function [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] 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 [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Similarly, Pechacek et al. reported deficits in motor impulsivity and attention following head injury were unaffected by minocycline treatment [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], raising questions about the efficacy of minocycline for the treatment of psychiatric disorders following head injury.\u003c/p\u003e \u003cp\u003eThe 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 [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Daily IP injections of 50 mg/kg of minocycline over four days reduced the biomarkers of inflammation and mitigated the behavioral deficits.\u003c/p\u003e \u003cp\u003eFrom 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 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Homsi et al provided the first evidence that a specific treatment regimen of minocycline could reduce brain edema in mice following head injury [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEdema makes a significant contribution to the neuropathology of head injury [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. 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[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. 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.\u003c/p\u003e \u003cp\u003eIn 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 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. 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.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e, 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 [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. 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[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eData Interpretation and Limitations\u003c/h2\u003e \u003cp\u003eThe 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\u0026ndash;4 subjects and may have been underpowered.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSummary\u003c/h2\u003e \u003cp\u003eA 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 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], a view shared by others to account for the many failed clinical trials for TBI [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. 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 \u0026ldquo;bump on the head\u0026rdquo; from the edema on the skin overlying the skull as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003e. This model, as reported in other studies on mild head injury [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], 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 [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. 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.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e: The CFF has a financial interest in Animal Imaging Research, a company that makes the radiofrequency electronics and holders for awake animal imaging. The CFF and PK have a partnership interest in Ekam Solutions the company that develops 3D MRI atlases for animal research.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; Contributions:\u003cbr\u003e\u0026nbsp;\u003c/strong\u003eAll of the authors have contributed substantially to the manuscript. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003eConcept, drafting and interpretation \u0026ndash; Hightower, Ferris, Kulkarni,\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eExecution and analysis \u0026ndash; Hightower, Prom, Brengel.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis work was supported by Elam Imaging, Inc. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e\u0026nbsp; We owe a dept of gratitude to Dr. Andrew Schafer whose scholarship supported the training and research of Ms. Rosemarie Hightower, an undergraduate student enrolled in the College of Science, Northeastern University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e: \u0026nbsp;All data available on request\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBrown R, Tozer D, Loubiere L, Hong Y, Fryer T, Williams G, Graves M, Aigbirhio F, O\u0026apos;Brien J, Markus H (2022) MINocyclinE to Reduce inflammation and blood brain barrier leakage in small Vessel diseAse (MINERVA) trial study protocol. 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Cells 10: Doi 10.3390/cells101026\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 3 is not available with this version\n\n\u003cp\u003eTables 1 to 2 are available in the Supplementary Files section\u003c/p\u003e"},{"header":"Supplementary Files ","content":"\u003cp\u003eSupplementary Files S5-S6 are not available with this version"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"translational-medicine-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trmc","sideBox":"Learn more about [Translational Medicine Communications](http://transmedcomms.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/TRMC/default.aspx","title":"Translational Medicine Communications","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"concussion, closed head momentum exchange, microgliosis, diffusion weighted imaging, cytotoxic edema","lastPublishedDoi":"10.21203/rs.3.rs-4228869/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4228869/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMinocycline is being tested in clinical trials for the treatment of stroke. As an antibiotic it reduces microglia activation. Can minocycline be used to treat mild head injury? \u0026nbsp;To that end, minocycline was tested in a novel, closed-head, momentum exchange model of repetitive mild head injury in female rats impacted while fully awake. MRI revealed there was no brain damage or contusion attesting to the mild nature of the head impacts in this model. It was hypothesized that drug treatment would reduce edema and brain neuroinflammation. \u0026nbsp;Female rats maintained on a reverse light-dark cycle were head impacted three times while fully awake with and without drug treatment. The impacts, separated by 24 hrs each, were delivered under red light illumination. Within 1-2 hrs of the last impact, rats were assessed for changes in water diffusion using diffusion weighted imaging. The data were registered to a 3D MRI rat atlas with 173 segmented brain areas providing site specific information\u003cstrong\u003e \u003c/strong\u003eon altered brain gray matter microarchitecture. Postmortem histology was performed 18 days post head injury. Head injury without minocycline treatment was characterized by multiple areas of increased fractional anisotropy, evidence of cytotoxic edema. Treatment with minocycline reversed these measures in many of the same areas and several others (e.g., hippocampus, basal ganglia, prefrontal cortex, sensory and motor cortices and thalamus). Histology for gliosis showed no evidence of neuroinflammation in the thalamus, hippocampus and cerebellum for control or experimental groups in this female model of mild head injury. These studies provide clear evidence that treatment with minocycline within hours after mild repetitive head injury significantly reduce measures of cytotoxic edema in a female rat model of mild repetitive head injury.\u003c/p\u003e","manuscriptTitle":"Testing the efficacy of minocycline treatment in an awake, female rat model of repetitive mild head injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-23 16:07:54","doi":"10.21203/rs.3.rs-4228869/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-04-17T15:46:22+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-17T05:25:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-09T06:17:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Translational Medicine Communications","date":"2024-04-08T04:35:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"translational-medicine-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trmc","sideBox":"Learn more about [Translational Medicine Communications](http://transmedcomms.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/TRMC/default.aspx","title":"Translational Medicine Communications","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"48d5015b-6229-426e-bcc8-f172d64df8db","owner":[],"postedDate":"April 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-04-23T16:07:54+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-23 16:07:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4228869","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4228869","identity":"rs-4228869","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}