Mild Traumatic Brain Injury with Associated Visual System Dysfunction: Investigating Histopathology, Functional Correlates, and a Novel Therapeutic Immune Modulator

Background. Traumatic brain injury (TBI) is a significant source of morbidity and mortality worldwide. Injuries associated with moderate to severe TBI can be profound, and have historically overshadowed the significant impact mild TBI (mTBI) can have on the lives of affected individuals. Mild TBI can manifest in a number of different ways, but one of the most significant and often debilitating is its impact on the visual system. In order to further investigate the underlying pathology of mTBI and test potential therapeutics, we developed a mouse model of mTBI induced by blast overpressure. In this model, a 50-60 psi blast wave from a highly pressurized bolus of air is directed at a focal region of the left lateral cranium of a mouse, and produces replicable motor, emotional, and visual system deficits with concomitant histopathology. Importantly, this model closely simulates functional visual system damage seen in human cases of mTBI. A major component of the brain’s reaction to trauma is an immune response that can cause additional long-term damage above and beyond that of the initial injury. This response was observed in our model as regions of microglial cell activation throughout areas of the brain important for visual processing. A novel therapeutic drug acting at cannabinoid type 2 receptors (CB2), known as SMM-189, had previously shown promise in improving visual outcome after mTBI in our model, but no studies were done to elucidate the cause of this improvement. The purpose of this dissertation was to further characterize visual system dysfunction and histopathology in our model, as well as investigate how the drug SMM-189 acts to exert its beneficial effects on these areas. Mice were blasted with either 50-psi or sham blast, and then injected over the next two weeks with either drug or vehicle intraperitoneally. Functional tests were administered at 30 days after blast, and perfused tissues were used for subsequent histologic evaluation. Tissue used for histologic analysis was collected from mice at 3 and 7 days post-blast, and in another cohort at 11 weeks after blast. Functional results. Optokinetic testing was administered to obtain visual acuity (VA) and contrast sensitivity (CS) thresholds in mice at 30 days after blast. It was found that no group showed any defects in VA, but the 50-psi vehicle-treated group (50V) showed significant deficits in the CS function of both eyes, which was completely rescued with drug treatment. Electroretinograms were run both pre- and post-blast on mice to obtain an electrophysiological readout of retinal cellular function over the first month after blast. The left eye of 50V animals showed a pathologic B-wave elevation, but no change in the A-wave, or peak latency times. Drug treatment corrected this abnormality, returning the 50-psi SMM-189 treatment group (50SMM) B-wave average back to control levels. Structural results. Optical coherence tomography at 30 days post-blast revealed pathologic outer retinal thinning in the left eye of 50V animals, with 50SMM animals showing no such change. Immunohistochemistry (IHC) to visualize microglia in the retina showed a significant microglial increase in the left eye of 50V animals at 3 days post-blast, and a lesser but still pathologic elevation in both left and right eyes at 30 days post-blast. Drug treatment decreased the pathologic microglial elevation at both 3 days and 30 days, indicating its efficacy in quelling inflammatory microglial recruitment. Another readout of pathological response in the retina, GFAP immunoreactivity, was found to be elevated in the left eye of 50V animals at 30 days as well. 50SMM animals did not show any GFAP immunoreactivity at this time point. Brn3a+ RGCs in the retina were visualized using IHC, and no significant changes were seen. Cross-sections through optic nerves (ON) were analyzed from animals 11 weeks after blast. Left ONs from both 50V and 50SMM animals were found to be atrophic compared to controls, while the right eyes were all equivalent. Manual axon counts revealed left ONs from 50V animals had a decreased axon density, as well as a decrease in total axon count. Animals in the 50SMM group had a decreased axon density in the left eye, but the total axon count returned to normal. The right ON of 50V animals also had a decrease in axon density, but the total axon count was not significantly different than controls. In the mouse brain, the right optic tract (ROT) contains predominantly the uncrossed axons originating from the left eye and optic nerve. The ROT of 50V animals at 3 days after blast showed a significant number of pathologic axon bulbs, indicating areas of traumatic axonal disruption. These tracts also showed an increased presence of M1 inflammatory-polarized microglia when compared to controls, as determined by IHC markers specific to the M1 polarization state. In the ROT of 50V animals at 5 and 7 days, large axon bulbs had decreased in number and numerous smaller granular accumulations became apparent, possibly indicating axonal degeneration. Drug treated animals showed a significant decrease in the number of axon bulbs at 3 days post-blast, and 20% of the microglia in this same tract had been converted from M1 to an M2 anti-inflammatory polarization state. Conclusion. The novel drug SMM-189 was shown to significantly improve many aspects of visual system damage in our model. Histologic evidence supports its role in positively modulating the immune response in neurotrauma, and acting to alter microglial polarization into a more neuroprotective phenotype. Furthermore, histologic benefits were associated with corresponding improvements in visual system function, showing its efficacy in treating mTBI visual system damage, a disease with no currently available pharmacotherapy. Future studies into mTBI-associated visual dysfunction should seek to investigate long-term outcome in this model, and to determine if drug benefit is sustained over an extended period after injury

Mild Traumatic Brain Injury Produces Neuron Loss That Can Be Rescued by Modulating Microglial Activation Using a CB2 Receptor Inverse Agonist

We have previously reported that mild TBI created by focal left-side cranial blast in mice produces widespread axonal injury, microglial activation, and a variety of functional deficits. We have also shown that these functional deficits are reduced by targeting microglia through their cannabinoid type-2 (CB2) receptors using two-week daily administration of the CB2 inverse agonist SMM-189. CB2 inverse agonists stabilize the G-protein coupled CB2 receptor in an inactive conformation, leading to increased phosphorylation and nuclear translocation of the cAMP response element binding protein (CREB), and thus bias activated microglia from a pro-inflammatory M1 to a pro-healing M2 state. In the present study, we showed that SMM-189 boosts nuclear pCREB levels in microglia in several brain regions by 3 days after TBI, by using pCREB/CD68 double immunofluorescent labeling. Next, to better understand the basis of motor deficits and increased fearfulness after TBI, we used unbiased stereological methods to characterize neuronal loss in cortex, striatum, and basolateral amygdala (BLA) and assessed how neuronal loss was affected by SMM-189 treatment. Our stereological neuron counts revealed a 20% reduction in cortical and 30% reduction in striatal neurons bilaterally at 2-3 months post blast, with SMM-189 yielding about 50% rescue. Loss of BLA neurons was restricted to the blast side, with 33% of Thy1+ fear-suppressing pyramidal neurons and 47% of fear-suppressing parvalbuminergic (PARV) interneurons lost, and Thy1-negative fear-promoting pyramidal neurons not significantly affected. SMM-189 yielded 50-60% rescue of Thy1+ and PARV neuron loss in BLA. Thus, fearfulness after mild TBI may result from the loss of fear-suppressing neuron types in BLA, and SMM-189 may reduce fearfulness by their rescue. Overall, our findings indicate that SMM-189 rescues damaged neurons and thereby alleviates functional deficits resulting from TBI, apparently by selectively modulating microglia to the beneficial M2 state. CB2 inverse agonists thus represent a promising therapeutic approach for mitigating neuroinflammation and neurodegeneration

A Novel Closed-Head Model of Mild Traumatic Brain Injury Using Focal Primary Overpressure Blast to the Cranium in Mice

Mild traumatic brain injury (TBI) from focal head impact is the most common form of TBI in humans. Animal models, however, typically use direct impact to the exposed dura or skull, or blast to the entire head. We present a detailed characterization of a novel overpressure blast system to create focal closed-head mild TBI in mice. A high-pressure air pulse limited to a 7.5 mm diameter area on the left side of the head overlying the forebrain is delivered to anesthetized mice. The mouse eyes and ears are shielded, and its head and body are cushioned to minimize movement. This approach creates mild TBI by a pressure wave that acts on the brain, with minimal accompanying head acceleration-deceleration. A single 20-psi blast yields no functional deficits or brain injury, while a single 25-40 psi blast yields only slight motor deficits and brain damage. By contrast, a single 50-60 psi blast produces significant visual, motor, and neuropsychiatric impairments and axonal damage and microglial activation in major fiber tracts, but no contusive brain injury. This model thus reproduces the widespread axonal injury and functional impairments characteristic of closed-head mild TBI, without the complications of systemic or ocular blast effects or head acceleration that typically occur in other blast or impact models of closed-skull mild TBI. Accordingly, our model provides a simple way to examine the biomechanics, pathophysiology, and functional deficits that result from TBI and can serve as a reliable platform for testing therapies that reduce brain pathology and deficits

Motor, Visual and Emotional Deficits in Mice after Closed-Head Mild Traumatic Brain Injury Are Alleviated by the Novel CB2 Inverse Agonist SMM-189

We have developed a focal blast model of closed-head mild traumatic brain injury (TBI) in mice. As true for individuals that have experienced mild TBI, mice subjected to 50–60 psi blast show motor, visual and emotional deficits, diffuse axonal injury and microglial activation, but no overt neuron loss. Because microglial activation can worsen brain damage after a concussive event and because microglia can be modulated by their cannabinoid type 2 receptors (CB2), we evaluated the effectiveness of the novel CB2 receptor inverse agonist SMM-189 in altering microglial activation and mitigating deficits after mild TBI. In vitro analysis indicated that SMM-189 converted human microglia from the pro-inflammatory M1 phenotype to the pro-healing M2 phenotype. Studies in mice showed that daily administration of SMM-189 for two weeks beginning shortly after blast greatly reduced the motor, visual, and emotional deficits otherwise evident after 50–60 psi blasts, and prevented brain injury that may contribute to these deficits. Our results suggest that treatment with the CB2 inverse agonist SMM-189 after a mild TBI event can reduce its adverse consequences by beneficially modulating microglial activation. These findings recommend further evaluation of CB2 inverse agonists as a novel therapeutic approach for treating mild TBI