Amelioration of visual deficits and visual system pathology after mild TBI with the cannabinoid type-2 receptor inverse agonist SMM-189.

Mild TBI is often accompanied by visual system dysfunction and injury, which is at least partly caused by microglial neuroinflammatory processes initiated by the injury. Using our focal cranial blast mouse model of closed-skull mild TBI, we evaluated the ability of the cannabinoid type-2 (CB2) receptor inverse agonist SMM-189, which biases microglia from the harmful M1 state to the beneficial M2 state, to mitigate visual system dysfunction and injury after TBI. Male C57BL/6 or Thy1-EYFP reporter mice received a closed-head blast of either 0-psi (sham) or 50-psi to the left side of the cranium. Blast mice received vehicle or 6 mg/kg SMM-189 daily beginning 2 h after blast. Sham mice received vehicle. In some mice, retina and optic nerve/tract were assessed morphologically at 3-7 days after blast, while other mice were assessed functionally by Optomotry 30 days after blast and morphologically at ≥30 days after blast. Mice sacrificed at 3-7 days were treated daily until sacrificed, while those assessed ≥30 days after blast were treated daily for 2 weeks post blast. Axon damage was evident in the left optic nerve and its continuation as the right optic tract at 3 days post blast in vehicle-treated blast mice in the form of swollen axon bulbs, and was accompanied by a significant increase in the abundance of microglia. Testing at 30 days post blast revealed that the contrast sensitivity function was significantly reduced in both eyes in vehicle-treated blast mice compared to vehicle-treated sham blast mice, and axon counts at ≥30 days after blast revealed a ∼10% loss in left optic nerve in vehicle-treated blast mice. Left optic nerve axon loss was highly correlated with the left eye deficit in contrast sensitivity. Immunolabeling at 30 days post blast showed a significant increase in the abundance of microglia in the retinas of both eyes and in GFAP + Müller cell processes traversing the inner plexiform layer in the left eye of vehicle-treated blast mice. SMM-189 treatment reduced axon injury and microglial abundance at 3 days, and mitigated axon loss, contrast sensitivity deficits, microglial abundance, and Müller cell GFAP upregulation at ≥30 days after blast injury. Analysis of right optic tract microglia at 3 days post blast for M1 versus M2 markers revealed that SMM-189 biased microglia toward the M2 state, with this action of SMM-189 being linked to reduced axonal injury. Taken together, our results show that focal left side cranial blast resulted in impaired contrast sensitivity and retinal pathology bilaterally and optic nerve loss ipsilaterally. The novel cannabinoid drug SMM-189 significantly mitigated the functional deficit and the associated pathologies. Our findings suggest the value of combatting visual system injury after TBI by using CB2 inverse agonists such as SMM-189, which appear to target microglia and bias them away from the pro-inflammatory M1 state, toward the protective M2 state.

Abnormalities in Dynamic Brain Activity Caused by Mild Traumatic Brain Injury Are Partially Rescued by the Cannabinoid Type-2 Receptor Inverse Agonist SMM-189.

Mild traumatic brain injury (mTBI) can cause severe long-term cognitive and emotional deficits, including impaired memory, depression, and persevering fear, but the neuropathological basis of these deficits is uncertain. As medial prefrontal cortex (mPFC) and hippocampus play important roles in memory and emotion, we used multi-site, multi-electrode recordings of oscillatory neuronal activity in local field potentials (LFPs) in awake, head-fixed mice to determine if the functioning of these regions was abnormal after mTBI, using a closed-skull focal cranial blast model. We evaluated mPFC, hippocampus CA1, and primary somatosensory/visual cortical areas (S1/V1). Although mTBI did not alter the power of oscillations, it did cause increased coherence of θ (4-10 Hz) and ß (10-30 Hz) oscillations within mPFC and S1/V1, reduced CA1 sharp-wave ripple (SWR)-evoked LFP activity in mPFC, downshifted SWR frequencies in CA1, and enhanced θ-γ phase-amplitude coupling (PAC) within mPFC. These abnormalities might be linked to the impaired memory, depression, and persevering fear seen after mTBI. Treatment with the cannabinoid type-2 (CB2) receptor inverse agonist SMM-189 has been shown to mitigate functional deficits and neuronal injury after mTBI in mice. We found that SMM-189 also reversed most of the observed neurophysiological abnormalities. This neurophysiological rescue is likely to stem from the previously reported reduction in neuron loss and/or the preservation of neuronal function and connectivity resulting from SMM-189 treatment, which appears to stem from the biasing of microglia from the proinflammatory M1 state to the prohealing M2 state by SMM-189.

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 2-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.

Confocal laser scanning microscopy and ultrastructural study of VGLUT2 thalamic input to striatal projection neurons in rats.

We examined thalamic input to striatum in rats using immunolabeling for the vesicular glutamate transporter (VGLUT2). Double immunofluorescence viewed with confocal laser scanning microscopy (CLSM) revealed that VGLUT2+ terminals are distinct from VGLUT1+ terminals. CLSM of Phaseolus vulgaris-leucoagglutinin (PHAL)-labeled cortical or thalamic terminals revealed that VGLUT2 is rare in corticostriatal terminals but nearly always present in thalamostriatal terminals. Electron microscopy revealed that VGLUT2+ terminals made up 39.4% of excitatory terminals in striatum (with VGLUT1+ corticostriatal terminals constituting the rest), and 66.8% of VGLUT2+ terminals synapsed on spines and the remainder on dendrites. VGLUT2+ axospinous terminals had a mean diameter of 0.624 µm, while VGLUT2+ axodendritic terminals a mean diameter of 0.698 µm. In tissue in which we simultaneously immunolabeled thalamostriatal terminals for VGLUT2 and striatal neurons for D1 (with about half of spines immunolabeled for D1), 54.6% of VGLUT2+ terminals targeted D1+ spines (i.e., direct pathway striatal neurons), and 37.3% of D1+ spines received VGLUT2+ synaptic contacts. By contrast, 45.4% of VGLUT2+ terminals targeted D1-negative spines (i.e., indirect pathway striatal neurons), and only 25.8% of D1-negative spines received VGLUT2+ synaptic contacts. Similarly, among VGLUT2+ axodendritic synaptic terminals, 59.1% contacted D1+ dendrites, and 40.9% contacted D1-negative dendrites. VGLUT2+ terminals on D1+ spines and dendrites tended to be slightly smaller than those on D1-negative spines and dendrites. Thus, thalamostriatal terminals contact both direct and indirect pathway striatal neurons, with a slight preference for direct. These results are consistent with physiological studies indicating slightly different effects of thalamic input on the two types of striatal projection neurons.