Date of Award
Doctor of Philosophy (PhD)
Anton J. Reiner, Ph.D.
Scott A. Heldt, Ph.D. Marcia G. Honig, Ph.D. Monica M. Jablonski, Ph.D. Bob M. Moore II, Ph.D.
Cannabinoid, Immune modulator, Microglia, Neuroimmunology, Traumatic brain injury, Visual system
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.
Guley, Natalie M. (http://orcid.org/0000-0002-4427-1799), "Mild Traumatic Brain Injury with Associated Visual System Dysfunction: Investigating Histopathology, Functional Correlates, and a Novel Therapeutic Immune Modulator" (2016). Theses and Dissertations (ETD). Paper 387. http://dx.doi.org/10.21007/etd.cghs.2016.0406.