Raloxifene Mitigates Emotional Deficits after Mild Traumatic Brain Injury in Mice.

Persons with mild traumatic brain injury (TBI) often exhibit persistent emotional impairments, particularly depression, fearfulness, and anxiety, that significantly diminish quality of life. Studying these mood disorders in animal models of mild TBI can help provide insight into possible therapies. We have previously reported that mice show increased depression, fearfulness, and anxiety, as well as visual and motor deficits, after focal cranial blast and that treatment with the cannabinoid type 2 receptor (CB2) inverse agonist, SMM-189, reduces these deficits. We have further shown that raloxifene, which is U.S. Food and Drug Administration approved as an estrogen receptor modulator to treat osteoporosis, but also possesses CB2 inverse agonism, yields a similar benefit for visual deficits in this model of TBI. Here, we have extended our studies of raloxifene benefit and show that it similarly reverses depression, fearfulness, and anxiety after focal cranial blast TBI in mice, using standard assays of these behavioral end-points. These results indicate the potential of raloxifene in the broad rescue of deficits after mild TBI and support phase 2 efficacy testing in human clinical trials.

Raloxifene, a cannabinoid type-2 receptor inverse agonist, mitigates visual deficits and pathology and modulates microglia after ocular blast.

Visual deficits after ocular blast injury (OBI) are common, but pharmacological approaches to improve long-term outcomes have not been identified. Blast forces frequently damage the retina and optic nerves, and work on experimental animals has shown the pro-inflammatory actions of microglia can further exacerbate such injuries. Cannabinoid type-2 receptor (CB2) inverse agonists specifically target activated microglia, biasing them away from the harmful pro-inflammatory M1 state toward the helpful reparative M2 state. We previously found that treating mice with CB2 inverse agonists after traumatic brain injury, produced by either focal cranial air blast or dorsal cranial impact, greatly attenuated the visual deficits and pathology that otherwise resulted. Here we examined the consequences of single and repeat OBI and the benefit provided by raloxifene, an FDA-approved estrogen receptor drug that possesses noteworthy CB2 inverse agonism. After single OBI, although the amplitudes of the A- and B-waves of the electroretinogram and pupil light response appeared to be normal, the mice showed hints of deficits in contrast sensitivity and visual acuity, a trend toward optic nerve axon loss, and significantly increased light aversion, which were reversed by 2 weeks of daily treatment with raloxifene. Mice subjected to repeat OBI (5 blasts spaced 1 min apart), exhibited more severe visual deficits, including decreases in contrast sensitivity, visual acuity, the amplitudes of the A- and B-waves of the electroretinogram, light aversion, and resting pupil diameter (i.e. hyperconstriction), accompanied by the loss of photoreceptor cells and optic nerve axons, nearly all of which were mitigated by raloxifene. Interestingly, optic nerve axon abundance was strongly correlated with contrast sensitivity and visual acuity across all groups of experimental mice in the repeat OBI study, suggesting optic nerve axon loss with repeat OBI and its attenuation with raloxifene are associated with the extent of these two deficits while photoreceptor abundance was highly correlated with A-wave amplitude and resting pupil size, suggesting a prominent role for photoreceptors in these two deficits. Quantitative PCR (qPCR) showed levels of M1-type microglial markers (e.g. iNOS, IL1ß, TNFα, and CD32) in retina, optic nerve, and thalamus were increased 3 days after repeat OBI. With raloxifene treatment, the overall expression of M1 markers was more similar to that in sham mice. Raloxifene treatment was also associated with the elevation of IL10 transcripts in all three tissues compared to repeat OBI alone, but the results for the three other M2 microglial markers we examined were more varied. Taken together, the qPCR results suggest that raloxifene benefit for visual function and pathology was associated with a lessening of the pro-inflammatory actions of microglia. The benefit we find for raloxifene following OBI provides a strong basis for phase-2 efficacy testing in human clinical trials for treating ocular injury.

Mesenchymal stem cell secretome protects against oxidative stress-induced ocular blast visual pathologies.

Visual deficits are a common concern among subjects with head trauma. Stem cell therapies have gained recent attention in treating visual deficits following head trauma. Previously, we have shown that adipose-derived stem cell (ASC) concentrated conditioned medium (ASC-CCM), when delivered via an intravitreal route, yielded a significant improvement in vision accompanied by a decrease in retinal neuroinflammation in a focal cranial blast model that indirectly injures the retina. The purpose of the current study is to extend our previous studies to a direct ocular blast injury model to further establish the preclinical efficacy of ASC-CCM. Adult C57BL/6J mice were subjected to repetitive ocular blast injury (rOBI) of 25 psi to the left eye, followed by intravitreal delivery of ASC-CCM (∼200 ng protein/2 µl) or saline within 2-3 h. Visual function and histological changes were measured 4 weeks after injury and treatment. In vitro, Müller cells were used to evaluate the antioxidant effect of ASC-CCM. Visual acuity, contrast sensitivity, and b-wave amplitudes in rOBI mice receiving saline were significantly decreased compared with age-matched sham blast mice. Immunohistological analyses demonstrated a significant increase in glial fibrillary acidic protein (a retinal injury marker) in Müller cell processes, DNA/RNA damage, and nitrotyrosine (indicative of oxidative stress) in the retina, while qPCR analysis revealed a >2-fold increase in pro-inflammatory cytokines (TNF-α, ICAM1, and Ccl2) in the retina, as well as markers for microglia/macrophage activation (IL-1ß and CD86). Remarkably, rOBI mice that received ASC-CCM demonstrated a significant improvement in visual function compared to saline-treated rOBI mice, with visual acuity, contrast sensitivity, and b-wave amplitudes that were not different from those in sham mice. This improvement in visual function also was associated with a significant reduction in retinal GFAP, neuroinflammation markers, and oxidative stress compared to saline-treated rOBI mice. In vitro, Müller cells exposed to oxidative stress via increasing doses of hydrogen peroxide demonstrated decreased viability, increased GFAP mRNA expression, and reduced activity for the antioxidant catalase. On the other hand, oxidatively stressed Müller cells pre-incubated with ASC-CCM showed normalized GFAP, viability, and catalase activity. In conclusion, our study demonstrates that a single intravitreal injection of ASC-CCM in the rOBI can significantly rescue retinal injury and provide significant restoration of visual function. Our in vitro studies suggest that the antioxidant catalase may play a major role in the protective effects of ASC-CCM, uncovering yet another aspect of the multifaceted benefits of ASC secretome therapies in neurotrauma.

Raloxifene Modulates Microglia and Rescues Visual Deficits and Pathology After Impact Traumatic Brain Injury.

Mild traumatic brain injury (TBI) involves widespread axonal injury and activation of microglia, which initiates secondary processes that worsen the TBI outcome. The upregulation of cannabinoid type-2 receptors (CB2) when microglia become activated allows CB2-binding drugs to selectively target microglia. CB2 inverse agonists modulate activated microglia by shifting them away from the harmful pro-inflammatory M1 state toward the helpful reparative M2 state and thus can stem secondary injury cascades. We previously found that treatment with the CB2 inverse agonist SMM-189 after mild TBI in mice produced by focal cranial blast rescues visual deficits and the optic nerve axon loss that would otherwise result. We have further shown that raloxifene, which is Food and Drug Administration (FDA)-approved as an estrogen receptor modulator to treat osteoporosis, but also possesses CB2 inverse agonism, yields similar benefit in this TBI model through its modulation of microglia. As many different traumatic events produce TBI in humans, it is widely acknowledged that diverse animal models must be used in evaluating possible therapies. Here we examine the consequences of TBI created by blunt impact to the mouse head for visual function and associated pathologies and assess raloxifene benefit. We found that mice subjected to impact TBI exhibited decreases in contrast sensitivity and the B-wave of the electroretinogram, increases in light aversion and resting pupil diameter, and optic nerve axon loss, which were rescued by daily injection of raloxifene at 5 or 10 mg/ml for 2 weeks. Raloxifene treatment was associated with reduced M1 activation and/or enhanced M2 activation in retina, optic nerve, and optic tract after impact TBI. Our results suggest that the higher raloxifene dose, in particular, may be therapeutic for the optic nerve by enhancing the phagocytosis of axonal debris that would otherwise promote inflammation, thereby salvaging less damaged axons. Our current work, together with our prior studies, shows that microglial activation drives secondary injury processes after both impact and cranial blast TBI and raloxifene mitigates microglial activation and visual system injury in both cases. The results thus provide a strong basis for phase 2 human clinical trials evaluating raloxifene as a TBI therapy.

Adipose Tissue-Derived Mesenchymal Stem Cell Concentrated Conditioned Medium Alters the Expression Pattern of Glutamate Regulatory Proteins and Aquaporin-4 in the Retina after Mild Traumatic Brain Injury.

Concentrated conditioned media from adipose tissue-derived mesenchymal stem cells (ASC-CCM) show promise for retinal degenerative diseases. In this study, we hypothesized that ASC-CCM could rescue retinal damage and thereby improve visual function by acting through Müller glia in mild traumatic brain injury (mTBI). Adult C57Bl/6 mice were subjected to a 50-psi air pulse on the left side of the head, resulting in an mTBI. After blast injury, 1 µL (∼100 ng total protein) of human ASC-CCM was delivered intravitreally and followed up after 4 weeks for visual function assessed by electroretinogram and histopathological markers for Müller cell-related markers. Blast mice that received ASC-CCM, compared with blast mice that received saline, demonstrated a significant improvement in a- and b-wave response correlated with a 1.3-fold decrease in extracellular glutamate levels and a concomitant increase in glutamine synthetase (GS), as well as the glutamate transporter (GLAST) in Müller cells. Additionally, an increase in aquaporin-4 (AQP4) in Müller cells in blast mice received saline restored to normal levels in blast mice that received ASC-CCM. In vitro studies on rMC-1 Müller glia exposed to 100 ng/mL glutamate or RNA interference knockdown of GLAST expression mimicked the increased Müller cell glial fibrillary acidic protein (a marker of gliosis) seen with mTBI, and suggested that an increase in glutamate and/or a decrease in GLAST might contribute to the Müller cell activation in vivo. Taken together, our data suggest a novel neuroprotective role for ASC-CCM in the rescue of the visual deficits and pathologies of mTBI via restoration of Müller cell health.

TSG-6 in conditioned media from adipose mesenchymal stem cells protects against visual deficits in mild traumatic brain injury model through neurovascular modulation.

BACKGROUND: Retinal inflammation affecting the neurovascular unit may play a role in the development of visual deficits following mild traumatic brain injury (mTBI). We have shown that concentrated conditioned media from adipose tissue-derived mesenchymal stem cells (ASC-CCM) can limit retinal damage from blast injury and improve visual function. In this study, we addressed the hypothesis that TNFα-stimulated gene-6 (TSG-6), an anti-inflammatory protein released by mesenchymal cells, mediates the observed therapeutic potential of ASCs via neurovascular modulation. METHODS: About 12-week-old C57Bl/6 mice were subjected to 50-psi air pulse on the left side of the head overlying the forebrain resulting in an mTBI. Age-matched sham blast mice served as control. About 1 µl of ASC-CCM (siControl-ASC-CCM) or TSG-6 knockdown ASC-CCM (siTSG-6-ASC-CCM) was delivered intravitreally into both eyes. One month following injection, the ocular function was assessed followed by molecular and immunohistological analysis. In vitro, mouse microglial cells were used to evaluate the anti-inflammatory effect of ASC-CCM. Efficacy of ASC-CCM in normalizing retinal vascular permeability was assessed using trans-endothelial resistance (TER) and VE-cadherin expression in the presence of TNFα (1 ng/ml). RESULTS: We show that intravitreal injection of ASC-CCM (siControl-ASC-CCM) but not the TSG-6 knockdown ASC-CCM (siTSG-6-ASC-CCM) mitigates the loss of visual acuity and contrast sensitivity, retinal expression of genes associated with microglial and endothelial activation, and retinal GFAP immunoreactivity at 4 weeks after blast injury. In vitro, siControl-ASC-CCM but not the siTSG-6-ASC-CCM not only suppressed microglial activation and STAT3 phosphorylation but also protected against TNFα-induced endothelial permeability as measured by transendothelial electrical resistance and decreased STAT3 phosphorylation. CONCLUSIONS: Our findings suggest that ASCs respond to an inflammatory milieu by secreting higher levels of TSG-6 that mediates the resolution of the inflammatory cascade on multiple cell types and correlates with the therapeutic potency of the ASC-CCM. These results expand our understanding of innate mesenchymal cell function and confirm the importance of considering methods to increase the production of key analytes such as TSG-6 if mesenchymal stem cell secretome-derived biologics are to be developed as a treatment solution against the traumatic effects of blast injuries and other neurovascular inflammatory conditions of the retina.

Amelioration of visual deficits and visual system pathology after mild TBI via the cannabinoid Type-2 receptor inverse agonism of raloxifene.

Visual deficits after traumatic brain injury (TBI) are common, but interventions that limit the post-trauma impairments have not been identified. We have found that treatment with the cannabinoid type-2 receptor (CB2) inverse agonist SMM-189 for 2 weeks after closed-head blast TBI greatly attenuates the visual deficits and retinal pathology this otherwise produces in mice, by modulating the deleterious role of microglia in the injury process after trauma. SMM-189, however, has not yet been approved for human use. Raloxifene is an FDA-approved estrogen receptor drug that is used to treat osteoporosis, but it was recently found to also show noteworthy CB2 inverse agonism. In the current studies, we found that a high pressure air blast in the absence of raloxifene treatment yields deficits in visual acuity and contrast sensitivity, reductions in the A-wave and B-wave of the scotopic electroretinogram (ERG), light aversion, and increased pupil constriction to light. Raloxifene delivered daily for two weeks after blast at 5-10 mg/kg mitigates or eliminates these abnormalities (with the higher dose generally more effective). This functional rescue with raloxifene is accompanied by a biasing of microglia from the harmful M1 to the helpful M2 state, and reductions in retinal, optic nerve, and oculomotor nucleus pathology. We also found that raloxifene treatment is still effective even when delayed until 48 h after TBI, and that raloxifene benefit appears attributable to its CB2 inverse agonism rather than its estrogenic actions. Our studies show raloxifene is effective in treating visual injury after brain and/or eye trauma, and they provide basis for phase-2 efficacy testing in human clinical trials.

The expression of tyrosine hydroxylase and DARPP-32 in the house crow (Corvus splendens) brain.

Birds of the family Corvidae which includes diverse species such as crows, rooks, ravens, magpies, jays, and jackdaws are known for their amazing abilities at problem-solving. Since the catecholaminergic system, especially the neurotransmitter dopamine, plays a role in cognition, we decided to study the distribution of tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of catecholamines in the brain of house crows (Corvus splendens). We also studied the expression of DARPP-32 (dopamine and cAMP-regulated phosphoprotein), which is expressed in dopaminoceptive neurons. Our results demonstrated that as in other avian species, the expression of both TH and DARPP-32 was highest in the house crow striatum. The caudolateral nidopallium (NCL, the avian analogue of the mammalian prefrontal cortex) could be differentiated from the surrounding pallial regions based on a larger number of TH-positive "baskets" of fibers around neurons in this region and greater intensity of DARPP-32 staining in the neuropil in this region. House crows also possessed distinct nuclei in their brains which corresponded to song control regions in other songbirds. Whereas immunoreactivity for TH was higher in the vocal control region Area X compared to the surrounding MSt (medial striatum) in house crows, staining in RA and HVC was not as prominent. Furthermore, the arcopallial song control regions RA (nucleus robustus arcopallialis) and AId (intermediate arcopallium) were strikingly negative for DARPP-32 staining, in contrast to the surrounding arcopallium. Patterns of immunoreactivity for TH and DARPP-32 in "limbic" areas such as the hippocampus, septum, and extended amygdala have also been described.

Concentrated Conditioned Media from Adipose Tissue Derived Mesenchymal Stem Cells Mitigates Visual Deficits and Retinal Inflammation Following Mild Traumatic Brain Injury.

Blast concussions are a common injury sustained in military combat today. Inflammation due to microglial polarization can drive the development of visual defects following blast injuries. In this study, we assessed whether anti-inflammatory factors released by the mesenchymal stem cells derived from adipose tissue (adipose stem cells, ASC) can limit retinal tissue damage and improve visual function in a mouse model of visual deficits following mild traumatic brain injury. We show that intravitreal injection of 1 µL of ASC concentrated conditioned medium from cells pre-stimulated with inflammatory cytokines (ASC-CCM) mitigates loss of visual acuity and contrast sensitivity four weeks post blast injury. Moreover, blast mice showed increased retinal expression of genes associated with microglial activation and inflammation by molecular analyses, retinal glial fibrillary acidic protein (GFAP) immunoreactivity, and increased loss of ganglion cells. Interestingly, blast mice that received ASC-CCM improved in all parameters above. In vitro, ASC-CCM not only suppressed microglial activation but also protected against Tumor necrosis alpha (TNFα) induced endothelial permeability as measured by transendothelial electrical resistance. Biochemical and molecular analyses demonstrate TSG-6 is highly expressed in ASC-CCM from cells pre-stimulated with TNFα and IFNγ but not from unstimulated cells. Our findings suggest that ASC-CCM mitigates visual deficits of the blast injury through their anti-inflammatory properties on activated pro-inflammatory microglia and endothelial cells. A regenerative therapy for immediate delivery at the time of injury may provide a practical and cost-effective solution against the traumatic effects of blast injuries to the retina.

Age-related decline in VIP-positive parasympathetic nerve fibers in the human submacular choroid.

PURPOSE: An age-related decline in macular choroidal blood flow (ChBF) occurs in humans. Vasodilatory nerve fibers containing vasoactive intestinal polypeptide (VIP) innervate choroidal blood vessels. The current study was conducted to examine the possibility that an age-related loss of these fibers might occur in the submacular choroid in humans, and thus contribute to a decline in ChBF. METHODS: Macular choroid punches were collected from 35 healthy human donors ranging from 21 to 93 years of age. Choroidal samples were immunolabeled using anti-VIP and the peroxidase-antiperoxidase METHOD: VIP-positive nerve fiber abundance was quantified in up to 12 fields per punch. Fifty macular punches were analyzed, and results for eye pairs were averaged. Choroidal vessel diameter (ChVD) was measured for these same fields. The relationship between age and vessel diameter or VIP-positive fiber abundance was analyzed. Multivariate statistical models were generated correcting for gender, variables related to the tissue specimens, and potential procedural sources of variability. RESULTS: The fully adjusted multivariate models showed a significant age-related reduction in both the VIP-positive fiber abundance (P = 0.0003, adjusted R(2) = 0.51) and ChVD (P < 0.0001, adjusted R(2) = 0.63), with slopes of -0.45 and -0.19, respectively. Adjusting for the same variables, VIP-positive fiber abundance showed a significant direct correlation with ChVD. CONCLUSIONS: The results indicate a significant age-related decline in VIP-positive nerve fibers and vessel diameter in the submacular choroid in disease-free human donor eyes. These findings suggest that a decline in the neural control of ChBF and vessel diameter may explain the reductions in ChBF and its adaptive control observed clinically with aging.

Cellular localization of AMPA type glutamate receptor subunits in the basal ganglia of pigeons (Columba livia).

Corticostriatal and thalamostriatal projections utilize glutamate as a neurotransmitter in mammals and birds. The influence on striatum is mediated, in part, by ionotropic AMPA-type glutamate receptors, which are heteromers composed of GluR1-4 subunits. Although the cellular localization of AMPA-type subunits has been well characterized in mammalian basal ganglia, their localization in avian basal ganglia has not. We thus carried out light microscopic single- and double-label and electron microscopic single-label immunohistochemical studies of GluR1-4 distribution and cellular localization in pigeon basal ganglia. Single-label studies showed that the striatal neuropil is rich in GluR1, GluR2, and GluR2/3 immunolabeling, suggesting the localization of GluR1, GluR2 and/or GluR3 to the dendrites and spines of striatal projection neurons. Double-label studies and perikaryal size distribution determined from single-label material indicated that about 25% of enkephalinergic and 25% of substance P-containing striatal projection neuron perikarya contained GluR1, whereas GluR2 was present in about 75% of enkephalinergic neurons and all substance-P -containing neurons. The perikaryal size distribution for GluR2 compared to GluR2/3 suggested that enkephalinergic neurons might more commonly contain GluR3 than do substance P neurons. Parvalbuminergic and calretininergic striatal interneurons were rich in GluR1 and GluR4, a few cholinergic striatal interneurons possessed GluR2, but somatostatinergic striatal interneurons were devoid of all subunits. The projection neurons of globus pallidus all possessed GluR1, GluR2, GluR2/3 and GluR4 immunolabeling. Ultrastructural analysis of striatum revealed that GluR1 was preferentially localized to dendritic spines, whereas GluR2/3 was found in spines, dendrites, and perikarya. GluR2/3-rich spines were generally larger than GluR1 spines and more frequently possessed perforated post-synaptic densities. These results show that the diverse basal ganglia neuron types each display different combinations of AMPA subunit localization that shape their responses to excitatory input. For striatal projection neurons and parvalbuminergic interneurons, the combinations resemble those for the corresponding cell types in mammals, and thus their AMPA responses to glutamate are likely to be similar.

An immunohistochemical and pathway tracing study of the striatopallidal organization of area X in the male zebra finch.

Area X is a nucleus within songbird basal ganglia that is part of the anterior forebrain song learning circuit. It receives cortical song-related input and projects to the dorsolateral medial nucleus of thalamus (DLM). We carried out single- and double-labeled immunohistochemical and pathway tracing studies in male zebra finch to characterize the cellular organization and circuitry of area X. We found that 5.4% of area X neuronal perikarya are relatively large, possess aspiny dendrites, and are rich in the pallidal neuron/striatal interneuron marker Lys8-Asn9-neurotensin8-13 (LANT6). Many of these perikarya were found to project to the DLM, and their traits suggest that they are pallidal. Area X also contained several neuron types characteristic of the striatum, including interneurons co-containing LANT6 and the striatal interneuron marker parvalbumin (2% of area X neurons), interneurons containing parvalbumin but not LANT6 (4.8%), cholinergic interneurons (1.4%), and neurons containing the striatal spiny projection neuron marker dopamine- and adenosine 3',5'-monophosphate-regulated phosphoprotein (DARPP-32) (30%). Area X was rich in substance P (SP)-containing terminals, and many ended on area X neurons projecting to the DLM with the woolly fiber morphology characteristic of striatopallidal terminals. Although SP+ perikarya were not detected in area X, prior studies suggest it is likely that SP-synthesizing neurons are present and the source of the SP+ input to area X neurons projecting to the DLM. Area X was poor in enkephalinergic fibers and perikarya. The present data support the premise that area X contains both striatal and pallidal neurons, with the striatal neurons likely to include SP+ neurons that project to the pallidal neurons.

Localization of preganglionic neurons that innervate choroidal neurons of pterygopalatine ganglion.

PURPOSE: The pterygopalatine ganglion (PPG) receives preganglionic input from the superior salivatory nucleus (SSN) of the facial motor complex and is the main source of parasympathetic input to the choroid in mammals. The present study was undertaken to determine in rats the location and neurotransmitters of SSN neurons innervating those PPG neurons that target the choroid and to determine the location and neurotransmitters of the PPG choroidal neurons themselves. METHODS: Retrograde labeling from rat choroid using a fluorescent tracer, in combination with immunofluorescence labeling for nitric oxide synthase (NOS), vasoactive intestinal polypeptide (VIP), and choline acetyltransferase (ChAT), was used to characterize the location and neurotransmitters of choroidal PPG neurons. To identify SSN neurons that innervate the choroidal PPG neurons, the Bartha strain of the retrograde transneuronal tracer pseudorabies virus (PRV-Ba) was injected into rat choroid, and immunolabeling for NOS or ChAT was used to characterize their neurochemistry. RESULTS: Fluorescent retrograde labeling showed that PPG neurons projecting to the choroid contained NOS, VIP, and ChAT and were widely distributed in PPG and its preganglionic root, the greater petrosal nerve. SSN neurons were ChAT(+), and a subset of them was found to contain NOS. PRV-Ba transneuronal retrograde labeling revealed that choroidal preganglionic neurons were localized to the rostral medioventral part of the ipsilateral SSN. The choroidal SSN neurons were ChAT(+) and appeared largely to correspond to the NOS(+) neurons of the SSN. CONCLUSIONS: These results show that preganglionic neurons in rats that are presumed to regulate choroidal blood flow through the PPG reside within the rostral medioventral SSN, and that NOS is a marker for these SSN neurons.