Sphingolipid changes in mouse brain and plasma after mild traumatic brain injury at the acute phases.

BACKGROUND: Traumatic brain injury (TBI) causes neuroinflammation and can lead to long-term neurological dysfunction, even in cases of mild TBI (mTBI). Despite the substantial burden of this disease, the management of TBI is precluded by an incomplete understanding of its cellular mechanisms. Sphingolipids (SPL) and their metabolites have emerged as key orchestrators of biological processes related to tissue injury, neuroinflammation, and inflammation resolution. No study so far has investigated comprehensive sphingolipid profile changes immediately following TBI in animal models or human cases. In this study, sphingolipid metabolite composition was examined during the acute phases in brain tissue and plasma of mice following mTBI. METHODS: Wildtype mice were exposed to air-blast-mediated mTBI, with blast exposure set at 50-psi on the left cranium and 0-psi designated as Sham. Sphingolipid profile was analyzed in brain tissue and plasma during the acute phases of 1, 3, and 7 days post-TBI via liquid-chromatography-mass spectrometry. Simultaneously, gene expression of sphingolipid metabolic markers within brain tissue was analyzed using quantitative reverse transcription-polymerase chain reaction. Significance (P-values) was determined by non-parametric t-test (Mann-Whitney test) and by Tukey's correction for multiple comparisons. RESULTS: In post-TBI brain tissue, there was a significant elevation of 1) acid sphingomyelinase (aSMase) at 1- and 3-days, 2) neutral sphingomyelinase (nSMase) at 7-days, 3) ceramide-1-phosphate levels at 1 day, and 4) monohexosylceramide (MHC) and sphingosine at 7-days. Among individual species, the study found an increase in C18:0 and a decrease in C24:1 ceramides (Cer) at 1 day; an increase in C20:0 MHC at 3 days; decrease in MHC C18:0 and increase in MHC C24:1, sphingomyelins (SM) C18:0, and C24:0 at 7 days. Moreover, many sphingolipid metabolic genes were elevated at 1 day, followed by a reduction at 3 days and an absence at 7-days post-TBI. In post-TBI plasma, there was 1) a significant reduction in Cer and MHC C22:0, and an increase in MHC C16:0 at 1 day; 2) a very significant increase in long-chain Cer C24:1 accompanied by significant decreases in Cer C24:0 and C22:0 in MHC and SM at 3 days; and 3) a significant increase of C22:0 in all classes of SPL (Cer, MHC and SM) as well as a decrease in Cer C24:1, MHC C24:1 and MHC C24:0 at 7 days. CONCLUSIONS: Alterations in sphingolipid metabolite composition, particularly sphingomyelinases and short-chain ceramides, may contribute to the induction and regulation of neuroinflammatory events in the early stages of TBI, suggesting potential targets for novel diagnostic, prognostic, and therapeutic strategies in the future.

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, 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.

Progressive long-term spatial memory loss following repeat concussive and subconcussive brain injury in mice, associated with dorsal hippocampal neuron loss, microglial phenotype shift, and vascular abnormalities.

There is considerable concern about the long-term deleterious effects of repeat head trauma on cognition, but little is known about underlying mechanisms and pathology. To examine this, we delivered four air blasts to the left side of the mouse cranium, a week apart, with an intensity that causes deficits when delivered singly and considered "concussive," or an intensity that does not yield significant deficits when delivered singly and considered "subconcussive." Neither repeat concussive nor subconcussive blast produced spatial memory deficits at 4 months, but both yielded deficits at 14 months, and dorsal hippocampal neuron loss. Hierarchical cluster analysis of dorsal hippocampal microglia across the three groups based on morphology and expression of MHCII, CX3CR1, CD68 and IBA1 revealed five distinct phenotypes. Types 1A and 1B microglia were more common in sham mice, linked to better neuron survival and memory, and appeared mildly activated. By contrast, 2B and 2C microglia were more common in repeat concussive and subconcussive mice, linked to poorer neuron survival and memory, and characterized by low expression levels and attenuated processes, suggesting they were de-activated and dysfunctional. In addition, endothelial cells in repeat concussive mice exhibited reduced CD31 and eNOS expression, which was correlated with the prevalence of type 2B and 2C microglia. Our findings suggest that both repeat concussive and subconcussive head injury engender progressive pathogenic processes, possibly through sustained effects on microglia that over time lead to increased prevalence of dysfunctional microglia, adversely affecting neurons and blood vessels, and thereby driving neurodegeneration and memory decline.

Role of the superior salivatory nucleus in parasympathetic control of choroidal blood flow and in maintenance of retinal health.

The vasodilatory pterygopalatine ganglion (PPG) innervation of the choroid is under the control of preganglionic input from the superior salivatory nucleus (SSN), the parasympathetic portion of the facial motor nucleus. We sought to confirm that choroidal SSN drives a choroid-wide vasodilation and determine if such control is important for retinal health. To the former end, we found, using transscleral laser Doppler flowmetry, that electrical activation of choroidal SSN significantly increased choroidal blood flow (ChBF), at a variety of choroidal sites that included more posterior as well as more anterior ones. We further found that the increases in ChBF were significantly reduced by inhibition of neuronal nitric oxide synthase (nNOS), thus implicating nitrergic PPG terminals in the SSN-elicited ChBF increases. To evaluate the role of parasympathetic control of ChBF in maintaining retinal health, some rats received unilateral lesions of SSN, and were evaluated functionally and histologically. In eyes ipsilateral to choroidal SSN destruction, we found that the flash-evoked scotopic electroretinogram a-wave and b-wave peak amplitudes were both significantly reduced by 10 weeks post lesion. Choroidal baroregulation was evaluated in some of these rats, and found to be impaired in the low systemic arterial blood pressure (ABP) range where vasodilation normally serves to maintain stable ChBF. In retina ipsilateral to SSN destruction, the abundance of Müller cell processes immunolabeled for glial fibrillary acidic protein (GFAP) and GFAP message were significantly upregulated. Our studies indicate that the SSN-PPG circuit mediates parasympathetic vasodilation of choroid, which appears to contribute to ChBF baroregulation during low ABP. Our results further indicate that impairment in this adaptive mechanism results in retinal dysfunction and pathology within months of the ChBF disturbance, indicating its importance for retinal health.

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.

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.

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.

Neurochemistry and circuit organization of the lateral spiriform nucleus of birds: A uniquely nonmammalian direct pathway component of the basal ganglia.

We used diverse methods to characterize the role of avian lateral spiriform nucleus (SpL) in basal ganglia motor function. Connectivity analysis showed that SpL receives input from globus pallidus (GP), and the intrapeduncular nucleus (INP) located ventromedial to GP, whose neurons express numerous striatal markers. SpL-projecting GP neurons were large and aspiny, while SpL-projecting INP neurons were medium sized and spiny. Connectivity analysis further showed that SpL receives inputs from subthalamic nucleus (STN) and substantia nigra pars reticulata (SNr), and that the SNr also receives inputs from GP, INP, and STN. Neurochemical analysis showed that SpL neurons express ENK, GAD, and a variety of pallidal neuron markers, and receive GABAergic terminals, some of which also contain DARPP32, consistent with GP pallidal and INP striatal inputs. Connectivity and neurochemical analysis showed that the SpL input to tectum prominently ends on GABAA receptor-enriched tectobulbar neurons. Behavioral studies showed that lesions of SpL impair visuomotor behaviors involving tracking and pecking moving targets. Our results suggest that SpL modulates brainstem-projecting tectobulbar neurons in a manner comparable to the demonstrated influence of GP internus on motor thalamus and of SNr on tectobulbar neurons in mammals. Given published data in amphibians and reptiles, it seems likely the SpL circuit represents a major direct pathway-type circuit by which the basal ganglia exerts its motor influence in nonmammalian tetrapods. The present studies also show that avian striatum is divided into three spatially segregated territories with differing connectivity, a medial striato-nigral territory, a dorsolateral striato-GP territory, and the ventrolateral INP motor territory.

Striatal parvalbuminergic neurons are lost in Huntington's disease: implications for dystonia.

Although dystonia represents a major source of motor disability in Huntington's disease (HD), its pathophysiology remains unknown. Because recent animal studies indicate that loss of parvalbuminergic (PARV+) striatal interneurons can cause dystonia, we investigated if loss of PARV+ striatal interneurons occurs during human HD progression, and thus might contribute to dystonia in HD. We used immunolabeling to detect PARV+ interneurons in fixed sections, and corrected for disease-related striatal atrophy by expressing PARV+ interneuron counts in ratio to interneurons co-containing somatostatin and neuropeptide Y (whose numbers are unaffected in HD). At all symptomatic HD grades, PARV+ interneurons were reduced to less than 26% of normal abundance in rostral caudate. In putamen rostral to the level of globus pallidus, loss of PARV+ interneurons was more gradual, not dropping off to less than 20% of control until grade 2. Loss of PARV+ interneurons was even more gradual in motor putamen at globus pallidus levels, with no loss at grade 1, and steady grade-wise decline thereafter. A large decrease in striatal PARV+ interneurons, thus, occurs in HD with advancing disease grade, with regional variation in the loss per grade. Given the findings of animal studies and the grade-wise loss of PARV+ striatal interneurons in motor striatum in parallel with the grade-wise appearance and worsening of dystonia, our results raise the possibility that loss of PARV+ striatal interneurons is a contributor to dystonia in HD.

How smart dinosaurs?

This commentary discusses the main points made in Reiner's article on the prospect that some theropod dinosaurs could have given rise to a lineage that achieved a human level of intelligence, and those made in Herculano-Houzel's article on the potentially monkey-like numbers of neurons in the pallium of large theropods, and the implications of this for their intelligence.

Could theropod dinosaurs have evolved to a human level of intelligence?

Noting that some theropod dinosaurs had large brains, large grasping hands, and likely binocular vision, paleontologist Dale Russell suggested that a branch of these dinosaurs might have evolved to a human intelligence level, had dinosaurs not become extinct. I offer reasons why the likely pallial organization in dinosaurs would have made this improbable, based on four assumptions. First, it is assumed that achieving human intelligence requires evolving an equivalent of the about 200 functionally specialized cortical areas characteristic of humans. Second, it is assumed that dinosaurs had an avian nuclear type of pallial organization, in contrast to the mammalian cortical organization. Third, it is assumed that the interactions between the different neuron types making up an information processing unit within pallium are critical to its role in analyzing information. Finally, it is assumed that increasing axonal length between the neuron sets carrying out this operation impairs its efficacy. Based on these assumptions, I present two main reasons why dinosaur pallium might have been unable to add the equivalent of 200 efficiently functioning cortical areas. First, a nuclear pattern of pallial organization would require increasing distances between the neuron groups corresponding to the separate layers of any given mammalian cortical area, as more sets of nuclei equivalent to a cortical area are interposed between the existing sets, increasing axon length and thereby impairing processing efficiency. Second, because of its nuclear organization, dinosaur pallium could not reduce axon length by folding to bring adjacent areas closer together, as occurs in cerebral cortex.

Spatial and temporal requirements for huntingtin (Htt) in neuronal migration and survival during brain development.

Huntington's disease (HD), caused by an expanded triplet repeat in the huntingtin (Htt) gene, results in extensive neuropathology, but study of the Htt gene in CNS development through gene knockout is problematic as the knockout leads to embryonic lethality in mice. Here, we report that the knockdown of Htt expression in neuroepithelial cells of neocortex results in disturbed cell migration, reduced proliferation, and increased cell death that is relatively specific to early neural development. In the cerebellum, however, Htt knockdown results in cell death but not perturbed migration. The cell death phenotype in cortex can be partially reversed with co-knockdown of Casp9, indicating that mitochondria-mediated cell apoptotic processes are involved in the neuronal death. The timing of knockdown during early development is also an important variable. These results indicate a spatial and temporal requirement for Htt expression in neural development. Although it is uncertain whether the loss of wild-type huntingtin function contributes to pathogenesis in Huntington's disease, these results clearly contraindicate the use of nonspecific knockdown of Htt as a therapeutic measure in HD, particularly in utero.

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.

A Long-Term Safety and Efficacy Report on Intravitreal Delivery of Adipose Stem Cells and Secretome on Visual Deficits After Traumatic Brain Injury.

Purpose: We compared intravitreal injection of human adipose stem cell concentrated conditioned media (ASC-CCM) to injection of live ASCs for their long-term safety and effectiveness against the visual deficits of mild traumatic brain injury (mTBI). Methods: We first tested different intravitreal ASC doses for safety. Other C57BL/6 mice then received focal cranial blast mTBI and were injected with the safe ASC dose (1000 cells/eye), ASC-CCM (∼200 ng protein/eye), or saline solution. At five and 10 months after blast injury, visual, molecular, and histological assessments evaluated treatment efficacy. Histological evaluation of eyes and other organs at 10 months after blast injury assessed safety. Results: Human ASCs at 1000 cells/eye were found to be safe, with >10,000 cells causing retinal damage. Blast-injured mice showed significant vision deficits compared to sham blast mice up to 10 months. Blast mice receiving ASC or ASC-CCM showed improved vision at five months but marginal effects at 10 months, correlated with changes in glial fibrillary acidic protein and proinflammatory gene expression in retina. Immunostaining for human IgG failed to detect ASCs in retina. Peripheral organs examined histologically at 10 months after blast injury were normal. Conclusions: Intravitreal injection of ASCs or ASC-CCM is safe and effective against the visual deficits of mTBI. Considering the unimproved glial response and the risk of retinal damage with live cells, our studies suggest that ASC-CCM has better safety and effectiveness than live cells for the treatment of visual dysfunction in mTBI. Translational Relevance: This study demonstrates the safety and efficacy of mesenchymal stem cell-based therapeutics, supporting them for phase 1 clinical studies.

Progression of basal ganglia pathology in heterozygous Q175 knock-in Huntington's disease mice.

We used behavioral testing and morphological methods to detail the progression of basal ganglia neuron type-specific pathology and the deficits stemming from them in male heterozygous Q175 mice, compared to age-matched WT males. A rotarod deficit was not present in Q175 mice until 18 months, but increased open field turn rate (reflecting hyperkinesia) and open field anxiety were evident at 6 months. No loss of striatal neurons was seen out to 18 months, but ENK+ and DARPP32+ striatal perikarya were fewer by 6 months, due to diminished expression, with further decline by 18 months. No reduction in SP+ striatal perikarya or striatal interneurons was seen in Q175 mice at 18 months, but cholinergic interneurons showed dendrite attenuation by 6 months. Despite reduced ENK expression in indirect pathway striatal perikarya, ENK-immunostained terminals in globus pallidus externus (GPe) were more abundant at 6 months and remained so out to 18 months. Similarly, SP-immunostained terminals from striatal direct pathway neurons were more abundant in globus pallidus internus and substantia nigra at 6 months and remained so at 18 months. FoxP2+ arkypallidal GPe neurons and subthalamic nucleus neurons were lost by 18 months but not prototypical PARV+ GPe neurons or dopaminergic nigral neurons. Our results show that striatal projection neuron abnormalities and behavioral abnormalities reflecting them develop between 2 and 6 months of age in Q175 male heterozygotes, indicating early effects of the HD mutation. The striatal pathologies resemble those in human HD, but are less severe at 18 months than even in premanifest HD.

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.

Systemic Elevation of n-3 Polyunsaturated Fatty Acids (n-3-PUFA) Is Associated with Protection against Visual, Motor, and Emotional Deficits in Mice following Closed-Head Mild Traumatic Brain Injury.

Traumatic brain injury (TBI) causes neuroinflammation and neurodegeneration leading to various pathological complications such as motor and sensory (visual) deficits, cognitive impairment, and depression. N-3 polyunsaturated fatty acid (n-3 PUFA) containing lipids are known to be anti-inflammatory, whereas the sphingolipid, ceramide (Cer), is an inducer of neuroinflammation and degeneration. Using Fat1+-transgenic mice that contain elevated levels of systemic n-3 PUFA, we tested whether they are resistant to mild TBI-mediated sensory-motor and emotional deficits by subjecting Fat1-transgenic mice and their WT littermates to focal cranial air blast (50 psi) or sham blast (0 psi, control). We observed that visual function in WT mice was reduced significantly following TBI but not in Fat1+-blast animals. We also found Fat1+-blast mice were resistant to the decline in motor functions, depression, and fear-producing effects of blast, as well as the reduction in the area of oculomotor nucleus and increase in activated microglia in the optic tract in brain sections seen following blast in WT mice. Lipid and gene expression analyses confirmed an elevated level of the n-3 PUFA eicosapentaenoic acid (EPA) in the plasma and brain, blocking of TBI-mediated increase of Cer in the brain, and decrease in TBI-mediated induction of Cer biosynthetic and inflammatory gene expression in the brain of the Fat1+ mice. Our results demonstrate that suppression of ceramide biosynthesis and inflammatory factors in Fat1+-transgenic mice is associated with significant protection against the visual, motor, and emotional deficits caused by mild TBI. This study suggests that n-3 PUFA (especially, EPA) has a promising therapeutic role in preventing neurodegeneration after TBI.

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.

Choroidal blood flow compensation in rats for arterial blood pressure decreases is neuronal nitric oxide-dependent but compensation for arterial blood pressure increases is not.

Choroidal blood flow (ChBF) compensates for changes in arterial blood pressure (ABP) and thereby remains relatively stable within a +/-40 mmHg range of basal ABP in rabbits, humans and pigeons. In the present study, we investigated if ChBF can compensate for increases and decreases in ABP in rats. ChBF was continuously monitored using laser Doppler flowmetry in anesthetized rats, and ABP measured via the femoral artery. At multiple intervals over a 2-4 h period during which ABP varied freely, ChBF and ABP were sampled and the results compiled across rats. We found that ChBF remained near baseline over an ABP range from 40 mmHg above basal ABP (90-100 mmHg) to 40 mmHg below basal ABP, but largely followed ABP linearly below 60 mmHg. Choroidal vascular resistance increased linearly as BP increased above 100 mmHg, and decreased linearly as BP declined from basal to 60 mmHg, but resistance declined no further below 60 mmHg. Inhibition of nitric oxide (NO) formation by either a selective inhibitor of neuronal nitric oxide synthase (NOS) (N(omega)-propyl-L-arginine) or a nonselective inhibitor of both neuronal NOS and endothelial NOS (N(omega)-nitro-l-arginine methyl ester) did not affect compensation above 100 mmHg ABP, but did cause ChBF to linearly follow declines in BP below 90 mmHg. In NOS-inhibited rats, vascular resistance increased linearly with BP above 100 mmHg, but remained at baseline below 90 mmHg. These findings reveal that ChBF in rats, as in rabbits, humans and pigeons, compensates for rises and/or declines in arterial blood pressure so as to remain relatively stable within a physiological range of ABPs. The ChBF compensation for low ABP in rats is dependent on choroidal vasodilation caused by neuronal NO formation but not the compensation for elevated BP, implicating parasympathetic nervous system vasodilation in the ChBF compensation to low ABP.