A model of repetitive mild brain injury without symptoms – risk for Parkinson’s disease with aging?

Objectives To test the hypothesis that repetitive mild traumatic brain injury in early life may be a potential risk factor for Parkinson’s disease. Methods A closed-head momentum exchange model was used to produce one or three mild concussions in young adult male rats as compare to non-injured, age and weight-matched controls. Six-seven weeks post-injury, rats were studied for deficits in cognitive and motor function Changes in brain anatomy and function were evaluated through analysis of resting state functional connectivity, diffusion weighted imaging with quantitative anisotropy and immunohistochemistry for neuroinflammation. Results Head injuries occurred without skull fracture or signs of intracranial bleeding or contusion. There were no significant differences in cognitive or motors behaviors between experimental groups. Rats concussed three times showed altered diffusivity in white matter tracts, basal ganglia, central amygdala, brainstem, and cerebellum. With a single concussion, the affected areas were limited to the caudate/putamen and central amygdala. Disruption of functional connectivity was most pronounced with three concussions as the midbrain dopamine system, hippocampus and brainstem/cerebellum showed hypoconnectivity. The suprachiasmatic nucleus was isolated from all functional connections. Interestingly, rats exposed to one concussion showed enhanced functional connectivity (or hyperconnectivity) across brain sites, particularly between the olfactory system and the cerebellum. Immunostaining for microglia activation showed inflammation in striatum and substantia nigra with three concussions but not with one. Interpretation Neuroradiological and immunohistochemical evidence of altered brain structure and function, particularly in striatal and midbrain dopaminergic areas, persists long after mild repetitive head injury. These changes may be long lasting and serve as early biomarkers of neurodegeneration and risk for Parkinson’s disease with aging.


Introduction
Traumatic brain injuries (TBI) are responsible for over 2.8 million emergency room visits and 50,000 deaths in the United States each year 1 . Mild TBI is characterized as a negligible loss of consciousness with minimal neuropathology 2,3 and is estimated to account for 70-90% of all TBI cases 4, 5 . Mild TBI following a single incident is difficult to detect, and most cognitive and behavioral deficits usually resolve within weeks of the head injury, with few cases resulting in extended recovery time periods 6-9 . However, a more pernicious, long-lasting condition may arise when the brain is exposed to repeated incidents of mild TBI (rmTBI) 10 . Repeated mild TBI is associated with more severe and protracted cognitive, motor, and behavioral complications that may last for months and even years [11][12][13] . Even after the remission of symptoms, there is accumulating evidence of persistent brain injuries 14-18 that carry an increased risk of dementia, Alzheimer's disease, chronic traumatic encephalopathy, and Parkinson's disease 5, 19-26 .
The objective of this study was to use a momentum exchange model of repetitive head injury to produce neuropathology in a rat that would suggest risk for Parkinson's disease (PD) with aging. It was important to establishment a specific number of head impacts that are necessary and sufficient to cause subtle or no changes in behavior in the presence of clear neuroradiological evidence of altered brain structure and function. Following disease progression over the natural life span of a rat would require the use of non-invasive magnetic resonance imaging (MRI) protocols. To these ends, we used diffusion weighted imaging (DWI) with indices of anisotropy registered to a 3D MRI rat atlas and computational analysis to identify putative changes in gray matter microarchitecture across 171 brain areas in control and experimental rats concussed one and three times. In addition, we used resting state-functional connectivity (rsFC) to evaluate changes in global functional neural circuitry. These two MRI protocols were selected due to their clinical use in diagnosing and following the progression of rmTBI after remission of symptoms 14-18 as well as their utility in identifying biomarkers of neurodegenerative disease 27-31 . We used a momentum exchange model developed by the National Football League to study player concussions and designed for preclinical studies by Viano and coworkers to scale to the human experience 32 . The velocity of head movement and energy transfer was calculated and scaled to mimic a mild concussive injury in humans producing no skull fractures, prolonged loss of consciousness, or signs of intracranial bleeding, all of which are more common in moderate and severe TBI 33 . Also using LFP, Aungst et al., scheduled concussions at two-day intervals, with as many as three recurrent injuries all under 2.5% isoflurane anesthesia 36 . In still another example, Fidan et al., used the controlled cortical impact model under 2% isoflurane with one, two, or three mild concussions 24 hrs apart 37 . Based on these and other studies, we scheduled one (n = 13) or three (n = 9) concussive head impacts under 2% isoflurane anesthesia, with a 48 hr interval between each impact. Control rats (n = 9) were exposed to isoflurane anesthesia three times with 48 hr intervals to control for the effects of anesthesia. Between 6-7 weeks after head injury, all animals were tested for cognitive and motor behavior and then imaged. At the end of the experiment 12 rats were put under deep anesthesia with 5% isoflurane and the thoracic cavity opened and the heart transcardially perfused with 4% paraformaldehyde. These brains were harvested for immunohistochemistry. The remaining rats were euthanized with a combination of carbon dioxide asphyxiation until the cessation of respiration followed by thoracotomy.

Neuroimaging
Imaging sessions were conducted using a Bruker Biospec 7.0T/20-cm USR horizontal magnet (Bruker, Billerica, MA, USA) and a 20-G/cm 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 (Animal Imaging Research, Holden, Massachusetts).
The design of the restraining system included a padded head support obviating the need for ear bars helping to reduce animal discomfort while minimizing motion artifact. All rats were imaged under 1-2% isoflurane while keeping a respiratory rate of 40-50/min. At the beginning of each imaging session, a high-resolution anatomical data set was collected using the RARE were recorded so that they could be regressed out in a later step. Functional data were assessed for the presence of motion spikes. Any large motion spikes were identified and removed from the time-course signals. This filtering step was followed by slice timing correction from interleaved slice acquisition order. Head motion correction (six motion parameters) was carried out using the first volume as a reference image. Normalization was completed by registering functional data to the 3D MRI Rat Brain Atlas (© 2012 Ekam Solutions LLC, Boston, MA) using affine registration through DRAMMS. The 3D MRI Rat Brain Atlas containing 171 annotated brain regions was used for segmentation. Data are reported in 166 brain areas, as five regions in the brain atlas were excluded from analysis due to the large size of three brains. These brains fell slightly outside our imaging field of view and thus we did not get any signal from the extreme caudal tip of the cerebellum. Whole brains that contain all regions of interest are needed for analyses so rather than excluding the animals, we removed the brain sites across all animals.
After quality assurance, band-pass filtering (0.01Hz ~ 0.1Hz) was preformed to reduce lowfrequency drift effects and high-frequency physiological noise for each subject. The resulting images were further detrended and spatially smoothed (full width at half maximum = 0.8mm).
Finally, regressors comprised of motion outliers, the six motion parameters, the mean white matter, and cerebrospinal fluid time series were fed into general linear models for nuisance regression to remove unwanted effects.
The region-to-region functional connectivity method was performed in this study to measure the correlations in spontaneous BOLD fluctuations. A network is comprised of nodes and edges; nodes being the brain region of interest (ROI) and edges being the connections between regions. 166 nodes were defined using the ROIs segmented from our custom MRI RAT Brain Atlas. Voxel time series data were averaged in each node based on the residual images using the nuisance regression procedure. Pearson's correlation coefficients across all pairs of nodes (14535 pairs) were computed for each subject among all three groups to assess the interregional temporal correlations. The r-values (ranging from -1 to 1) were z-transformed using the Fisher's Z transform to improve normality. 166 x 166 symmetric connectivity matrices were constructed with each entry representing the strength of edge. Group-level analysis was performed to look at the functional connectivity in all experimental groups. The resulting Z-score matrices from one-group t-tests were clustered using the K-nearest neighbors clustering method to identify how nodes cluster together and form resting state networks. A Z-score threshold of |Z|=2.3 was applied to remove spurious or weak node connections for visualization purposes.

Behavioral Testing
The novel object recognition task (NOR) was used to assess episodic learning and memory 39, 40 . The apparatus consisted of a black cube-shaped Plexi-glass box (L:60.9 W: 69. A tapered balance beam (Dragonfly Inc., Ridgeley, WV) and the rota-rod were used to measure motor behavior as described 44,45 . The balance beam (L: 150 cm, W: 5.5 cm tapering down to 1.5 cm, elevated 120 cm) was equally divided into three sections (L:47 cm each; "wide", "middle", "thin" sections) that were lined with touch-sensitive sensor ledges (Width: 2 cm) that ran the length of the beam and were arranged on each side, 4 cm below the surface of the beam to count paw slips (or foot faults). At the start of the maze ("wide" section), was a wooden start platform, and at the end of the beam (immediately following the "thin" section) was a black enclosed Plexiglas goal box. After 2 days of training (3 trials per day), animals were tested (3 trials/day for 2 days). Prior to each trial, animals were placed inside the goal box for 1 min. Animals were then placed on a start platform and timed for traversing into the goal box, where they remained for 1 min, and were then placed back in their home cage until the next trial (30 min intertrial interval).
Following 2 days of training (3 trials/day), animals were tested over 2 days (3 trials/day) using the rota-rod by placing them on a rotating cylinder (diameter: 4 cm) that rotated at an increasing frequency starting at 1 rpm and increasing linearly at a 0.1 v/t2 acceleration rate for a total of 210 seconds ending at a max frequency of 50 rpm. Latency to fall off the rod was recorded for each animal and averaged across trials and days. For all behavior measures, GraphPad Prism version 6.0 (GraphPad Software, La Jolla, CA) was used for statistical analyses.
One-sample t-tests were assessed differences from chance levels (i.e., = 50%) of exploration in the NOR task, for each experimental group individually. Comparisons between groups were assessed using one-way analysis of variance (ANOVA) or mixed ANOVAs followed by Fisher's protected least significant difference post-hoc test.

Iba1 Immunohistochemistry
Rats (n=3 0-hit, n=5 1-hit, n=4 3-hit) were perfused with 4% paraformaldehyde. Brains Microwave-assisted immunohistochemistry was performed according to procedures described in Hoffman et al, 46 . The primary antibody is rabbit anti-Iba1 (Wako) used at a dilution of 1:10,000. Floating sections were incubated for 48hrs at 4 degrees C on a gyrotary shaker. Secondary antibody (biotinylateed goat anti-rabbit IgG) and ABC incubations were carried out according to instructions from the supplier Vector Laboratories and modified according to Hoffman et al. 46 Nickel-DAB was the chromagen; reagents were dissolved in 0.175M sodium acetate, pH 6.8. Hydrogen peroxide was added to a final concentration of .0025% to begin the histochemical reaction. The Ni-DAB reaction was stopped at 15 minutes by moving sections to 0.175M sodium acetate, and rinsing them several times in the same solution.
Immunoreactive cells and fibers appear blue/black. Sections were mounted on gelatin coated slides, dried overnight, stained in neutral red as a counterstain for the immunoreactivity, dehydrated in graded ethanols, cleared in Histoclear and coverslipped with Histomount. Sections were examined and photographed using a Nikon microscope equipped with a digital camera assisted by IVision image software. and three hit animals (t(11) = 6.84, p < 0.0001; t(9) = 3.86, p < 0.01; and t(7) = 4.9, p < 0.001, respectively) all had a significantly greater preference for the novel object, beyond chance (>50%) during the novel phase (See Fig. 1). Table 1 summarizes the results of locomotor testing showing no differences between the experimental groups.

Diffusion Weighted Imaging and Quantitative Anisotropy
Measures of anisotropy at 6-7 weeks post injury were registered to the 3D MRI Rat Atlas with 171 segmented brain areas to identify possible changes in gray matter microarchitecture 38 .
The data for fractional anisotropy (FA) and radial diffusivity (RD) are shown in Fig. 2. These probability heat maps show statistical differences between the one and three hit groups as compared to controls. The effects on FA from a single concussion were limited to the dorsal striatum and central amygdala. However, rats exposed to three concussions showed significant FA changes in the olfactory system, basal ganglia, central amygdala, cerebellum, and deep cerebellar nuclei. The RD changes with one concussion were limited to dorsal and ventral striatum, medial amygdala, and trigeminal nerve. RD changes with three concussions covered motor, somatosensory, and insular cortices; dorsal and ventral striatum, globus pallidus, and the superior colliculus.

Resting State Functional Connectivity
The delineated areas in the two correlation matrices in lobules) and deep cerebellar nuclei (lateral and interposed). In contrast, rats exposed to three concussions have reduced connectivity that is limited only to the olfactory bulb. The SCN, the key node in the brain controlling circadian rhythms and sleep/wake cycles, has functional connections with adjacent areas of the hypothalamus in control rats that are reduced with one hit and eliminated with three hits.
The ventral tegmental area (VTA) as well as the substantia nigra compacta (SNc) and reticularis (SNr) make up the core nodes of the midbrain dopaminergic system. From these regions, control animals have diffuse connectivity to areas in the amygdala, hypothalamus, thalamus, medulla oblongata, and cerebellum. Following a single concussion, the functional connectivity primarily coalesces around the thalamus. Animals exposed to repeated concussions showed reduced connectivity as compared to the other groups, and had no connectivity between the SN and the VTA.
The sensitivity of the cerebellum and its efferent connections to the brain through the deep cerebellar nuclei was examined further by seeding the combined lateral, fastigial, and interposed nuclei, and mapping areas of connectivity in the one and three hit groups that were significantly different from control (see Fig. 4). In addition, the posterior cerebellum was also seeded using an aggregate of multiple areas, (6-10 lobules, cupola, crus 1 and 2, paramedian, and paraflocculus). The purpose of this seeding strategy was to identify putative afferent connections to the posterior cerebellum given its enhanced functional connectivity following a single concussion. For the one hit group, there was strong connectivity with the olfactory bulb, prelimbic ctx, tenia tecta and endopiriform ctx (section E and F). The amygdala (central, medial and basal, section D), hippocampus (CA3 dorsal and ventral, CA1 dorsal, sections D and C), motor ctx (section D) and medulla oblongata (olivary n., vestibular n. principle sensory n. trigeminal, and parvicellular reticular n., sections A and B) all showed strong connectivity to the posterior cerebellum. These connections were fewer and less significant with repeated concussions. The reorganization of functional connectivity in the cerebellum and brainstem shown in Figures 3 and 4 compliment the FA data (Fig. 2) showing alterations in water diffusion and putative gray matter microarchitecture across many of the same brain areas.

Iba1 Immunohistochemistry
While the differences in neuroradiology data from DWI and rsFC were consistent between one and three hit cohorts as measured by the effect size and variance, the data from IHC showing sites of neuroinflammation were more variable. In non-concussed rats, microglia were evenly distributed with a similar appearance of smallish cell bodies, with 2-5 smooth fibers. The size, appearance, and density of Iba1 immunopositive cells in the 1-hit animals were variable. Three of the one hit rats exhibited bushy microglia with a bottle brush appearance throughout the brain as shown in cerebellum (Fig 7). One of the one hit rats showed patches of microglia activation in the periaqueductal gray of the midbrain and medulla (data not shown) and another one hit rat showed patches of activation in the striatum (Fig 5). These patches of intense Iba1 staining show microglia with large cell bodies and fat, short processes. The three hit rats showed large areas of neuroinflammation that were more dispersed along the neural axis. All three hit animals showed low to high intense staining of patches in the striatum (Fig 5) while two three hit rats showed intense microglia activation in SNr and VTA (Fig 6). The cerebellum showed no patches of Iba1 staining in one or three hit rats. However, they have a bushy, bottle brush appearance indicative of activation as shown in Fig 7. These bushy microglia were found in all cerebellar lobules for one and three hit rats.

Discussion
The purpose of our study was to develop a translational model of rmTBI that produced little if any overt behavioral deficits in the presence of altered brain organization and function that may pose a risk for neurodegenerative disease later in life. Indeed, we were unable to identify any changes in cognitive or motor function at six-seven weeks post injury in one or three hit rats. It is always possible that cognitive and motor deficits would have been revealed with different and more interrogative assays, but our assessment methods showed no overt problems with general health and behavior using this model of rmTBI. Nonetheless, noninvasive imaging using DWI and rsFC protocols and post mortem histology revealed significant alterations in putative gray matter architecture, functional connectivity and neuroinflammation in concussed rats with repetitive injury producing the greatest pathology. Several studies have reported alterations in cerebral glucose utilization following TBI in both humans and animals. The change in metabolism following injury is triphasic with an initial period of hyperglycolysis followed by depressed glucose metabolism and finally recovery 54-58 .

One vs Three Concussions
In a recent study, Selwyn and coworkers looked at repeated head injury in rodents at specific time periods determined to coincide with reduced glucose uptake, and reported greater neurological damage as well as deficits in motor function, thus corroborating earlier studies that the brain needs time to recover 56 . Concussions that occur closer together have greater cognitive and behavioral consequences, and have lasting deficits that can be present up to year later in preclinical models 56 . Relatedly, in a repeated imaging study Qin et al., showed that, at multiple time points both during and after repeated head strikes, FA and mean diffusivity, as well as axial and radial diffusivity continue to change over time across various regions of the brain 59 .

Diffusion Weighted Imaging
As noted above, the changes in indices of anisotropy at six-seven weeks following a single concussion were few. The changes that occurred were localized to the central and medial amygdala, and the dorsal/ventral striatum (caudate/putamen). These areas are related to the control of emotion and dopaminergic regulation of motor function, respectively 60-62 . Rats exposed to three concussions showed significant changes in FA within the olfactory system, basal ganglia, central amygdala, cerebellum, and deep cerebellar nuclei, while changes in RD were reflected in the motor, somatosensory cortices, basal ganglia, and superior colliculus. The resulting putative changes to gray matter microarchitecture show a distinct separation between forebrain and hindbrain (see 3D sagittal representation in Fig. 2). These results align with numerous reports showing that the cerebellum is particularly vulnerable to mild TBI 53, 63-67 .
Furthermore, in a recent study, rsFC data from human mTBI patients identified altered connectivity to the cerebellum as an important biomarker for detecting mTBI 14 . Related to this finding, data from retired military personnel show that decreased metabolic activity in the cerebellum is negatively correlated with the number of blast-related mild TBIs 63 . Given the heterogeneity of TBIs, the consistency of alterations to cerebellar function due to head injury in both humans and across animal models is uniquely distinct and suggests that the cerebellum is an important region for characterizing the progression of head injury.

Resting State Functional Connectivity: Suprachiasmatic Nucleus
This study included a global analysis of rsFC of 166 brain regions extending from the rostral-most portion of the olfactory bulb to the caudal brainstem and cerebellum. Animals concussed once showed a combination of hyper-and hypoconnectivity across several networks, while rats concussed three times presented with only hypoconnectivity (see Fig. 3). In both one and three hit animals, the SCN had injury-dependent hypoconnectivity as compared to nonconcussed rats that showed strong connectivity between the SCN and the medial basal hypothalamus. After a single hit, the connectivity contracted to a smaller cluster, and with three hits the SCN lost all connectivity with this network.
The SCN has a critical role in circadian timing and regulation of sleep-wake cycles.
Altered sleep is an important parameter in the development of our proposed animal model, as the prevalence of sleep disorders among TBI patients is approximately 50% [68][69][70] . Interestingly, sleep disturbance also often appears as an early symptom in both Alzheimer's and Parkinson's Disease [71][72][73][74] , and brain tissue from Alzheimer's patients show pronounced reductions in both SCN volume and neuron density 75,76 . This relates to data generated using the fluid percussion model that show that TBI disrupts SCN circadian gene expression 77 . Reduced SCN function coupled with altered sleep patterns across TBI and Alzheimer's may suggest that a similar mechanism underlies both conditions. For example, during the preclinical stages of Alzheimer's disease, increased amyloid-β (Aβ) accumulation coincides with the deterioration of sleep quality that, in turn, corresponds to cognitive decline and wakefulness 78 . Similarly, in TBI, increased Aβ deposition is present acutely 79,80 , and years after initial head trauma 81,82 . A growing body of research indicates that Aβ is removed from the brain through the glymphatic system that reaches peak functionality during sleep 83,84 . Thus, disrupted sleep reduces the effectiveness of the glymphatic system leading to increased brain Aβ. Together these studies suggest that TBI damage to the SCN and related sleep circuits can alter circadian timing causing an accumulation of Aβ that prehaps contributes to the risk of developing Alzheimer's disease later in life.
To our knowledge, the rsFC data is the first example showing an injury-dependent loss of connectivity in the SCN. To begin to examine the implications that this finding has for the pathogenesis of neurodegenerative disease, future studies using the momentum exchange model for rmTBI will investigate whether a loss of SCN connectivity corresponds to altered sleep patterns and/or other disturbances associated with circadian rhythms e.g., feeding, temperature, endocrine hormones.

Resting State Functional Connectivity: Midbrain Dopamine System
The connectivity of the midbrain dopaminergic system, germane to the development of

Resting State Functional Connectivity: Olfactory System/Cerebellum
One of the more interesting observations from rsFC is the relationship between the olfactory system and the cerebellum. Non-concussed rats showed the olfactory bulb and anterior olfactory n. have close adjacent connections to the orbital and piriform cortices. Six-seven weeks post injury, rats concussed only once showed increased functional connections in the forebrain olfactory system and limbic ctx with hindbrain regions that include the anterior cerebellum (3-5 lobules) and deep cerebellar nuclei (lateral and interposed). In stark contrast, rats exposed to three concussions had reduced connectivity limited only to the olfactory bulb and isolated from the anterior olfactory n. The extension of connectivity across the long axis of the brain between two seemingly disparate regions with a single insult was not initially hypothesized. Both the olfactory bulb and cerebellum have numerous polysynaptic connections to much of the brain 89-91 . However, their primary connection may be through the 5 th cranial nerve as the perception of many odors involves the interaction of olfactory bulbs and the trigeminal system 92 .
BOLD imaging in response to odors that involve both systems show brain activation in the olfactory cortex, insula, thalamus, and cerebellum 93 . Indeed, the cerebellum is consistently activated in human imaging studies that use an odor stimulus 94 . While the pathway from the olfactory bulbs to cerebellum has not yet been defined, the circuitry appears to cross over the midline as lesions in the left cerebellum impair odor processing in the contralateral nostril 94 .
Moreover, data based on changing odor intensity suggest that the intranasal trigeminal system may be responsible for odor-induced activation of the cerebellum 93, 95 . Disrupted olfaction is commonly found long after initial head trauma in TBI patients 96 , and is a highly prevalent and early symptom of Parkinson's and Alzheimer's disease 87, 97-99 . The differences in connectivity between the cerebellum and olfactory system with single and repeated concussions may underscore the importance of their relationship, and possibly identify novel markers for neurodegenerative disease following early head trauma.

Resting State Functional Connectivity: Cerebellum
The sensitivity of the cerebellum and its efferent connections to the brain through the cerebellar nuclei was examined by seeding the combined dentate, fastigial, and interposed nuclei as well as the posterior cerebellum, and then mapping their areas of connectivity. The cerebellum has reciprocal interactions with much of the brain 100

Considerations and Limitations
While behavioral assays revealed no significant differences between controls and concussed animals, it should be noted that testing occurred six-seven weeks post injury and thus, initial dysfunction may have been followed by recovery. It is generally held that the abatement of biopsychosocial deficits is accompanied by a parallel resolution of neuroradiological evidence of brain injury. While this is not the case in this study, in a recent set of experiments, Rajesh and coworkers reported that neural disruptions and structural insult in mTBI may persist up to 10 years following injury in subjects with normal cognitive function 17 . Hypoconnectivity in the forebrain proposed to be responsible for initial cognitive deficits persisted for years after injury suggesting the brain may compensate for disrupted function through reorganization. A time lapse of six-seven weeks in a rat's life is comparable to 4-5 years in humans, and thus the continued presence of injury after rmTBI suggests that the hyper-and hypoconnectivity and neuroinflammation observed in these studies may persist over the life of the rat.    Shown are correlation matrices of 166 rat brain areas for rsFC comparing controls to one concussion (top left) and three concussions (bottom left). Each dark red pixel for control rats represents one of 166 brain areas that is significantly correlated with other brain areas. The brain areas with significant correlations appear as clusters because they are contiguous in their neuroanatomy and function. The diagonal line separates the control and one concussed groups.
The pixels for one concussion are a mirror image of those pixels (i.e. brain areas for controls).
Interesting, rats with a single concussion (n=13) showed greater rsFC within the anterior and posterior cerebellum and deep cerebellar n., olfactory system and prefrontal ctx when compared to no hit controls (n = 9).

Figure 4. Seeding the cerebellum
The deep cerebellar nuclei (lateral, fastigial and interposed) and collective areas comprising the posterior cerebellum served as seeds point to focus on the connectivity of the brain to the cerebellum following one and three concussions (hits) as compared to 0 hit sham controls. Areas denoted in red/yellow are significantly greater than control while blue are significantly less than control. Sections A and B show increased connectivity between the deep cerebellar nuclei and the posterior cerebellum (lobules 5-9), crus 1 of the ansiform lobule, and paraflocculus in one hit rats. Additionally, the primary sensory n. of the trigeminal nerve, vestibular n., parvicellular reticular n., and the olivary n. of the underlying medulla oblongata are all part of the enhanced functional connectivity that is present in one hit rats as compared to control animals. These same cerebellar/ brain stem connections were reduced in the three hit rats (controls < three hit < one hit, sections A and B). At the level of the pons (section C) there is clear bilateral connectivity to the lateral part of the SN in one hit rats and, to a lesser degree, a unilateral connection in the three hit rats. Three hit rats show a reduced connectivity (blue) in the red n., dorsal CA1, and dorsal subiculum of the hippocampus (three hit < control) but an increased connectivity in the ventral CA1, visual 1 and 2 cortices (control < three hit). At the level of the thalamus (section D) there is only one area that differs between one hit and control animals, the retrosplenial ctx. In contrast, the three hit rats show enhanced bilateral connectivity in the ventral posterior thalamus and paracentral thalamic n., and reduced connectivity in dorsal CA3 hippocampus and piriform ctx. At the level of the striatum (section E) there is reduced connectivity in one and three hit rats in the accumbens shell (one hit ≤ three hit < control). Three hit rats show a bilateral reduction in the primary somatosensory cortex representing the jaw, a unilateral reduction in connectivity to the motor ctx, and enhanced connectivity in the piriform ctx and diagonal band of Broca.

Figure 5. Activated Microglia in Striatum
Shown are micrographs of axial sections of the striatum (caudate/putamen) of rats concussed one and three times as compared to non-concussed rats ( 0 hit). Microglia immunostained with Iba1 antibody appear as multipolar black labeled cells, of which the number and density is greatly increased with three hits.