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.

Formulation and performance evaluation of polymeric mixed micelles encapsulated with baicalein for breast cancer treatment.

The present study is aimed to formulate baicalein-loaded mixed micelles to enhance the solubility and oral bioavailability. Baicalein encapsulated D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) and pluronic F127 (F127) combined micelles were prepared and investigated for anticancer effect. The optimized formulation contains 25.04 ± 0.24 nm mean particle size of micelles with a zeta potential value of -4.01 ± 0.5 mV. The calculated entrapment efficiency percentage of baicalein within the micellar structure was 83.43 ± 0.13% and the in vitro release of baicalein from micelles displayed a sustained release profile at pH 7.4. The incorporation of baicalein within micelles core was also confirmed by FTIR analysis of formulation, which hardly represents the characteristic peak of baicalein, indicating successful entrapment of the drug. In vitro cell culture experiments revealed baicalein-loaded micelles significantly enhanced cellular uptake and cytotoxicity against MDAMB-231 cell lines in comparison to free baicalein. Additionally, as compared to free baicalein, baicalein micelles demonstrated greater apoptosis-inducing potential while the results of the cell cycle study exhibited arrest of cells at the G0/G1 phase of the cell cycle. Results of ROS (reactive oxygen species) and MMP (mitochondrial membrane potential) assay revealed the ROS-dependent mitochondrial-mediated apoptosis pathway utilized by developed formulation to inhibit cell proliferation. Thus, the developed nano micelles can serve as a potent carrier system for baicalein against breast cancer.

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.

Immunohistochemical Localization of GFAP and Glutamate Regulatory Proteins in Chick Retina and Their Levels of Expressions in Altered Photoperiods.

Moderate to intense light is reported to damage the chick retina, which is cone dominated. Light damage alters neurotransmitter pools, such as those of glutamate. Glutamate level in the retina is regulated by glutamate-aspartate transporter (GLAST) and glutamine synthetase (GS). We examined immunolocalization patterns and the expression levels of both markers and of glial fibrillary acidic protein (GFAP, a marker of neuronal stress) in chick retina exposed to 2000 lux under 12-h light:12-h dark (12L:12D; normal photoperiod), 18L:6D (prolonged photoperiod), and 24L:0D (constant light) at post-hatch day 30. Retinal damage (increased death of photoreceptors and inner retinal neurons and Müller cell hypertrophy) and GFAP expression in Müller cells were maximal in 24L:0D condition compared to that seen in 12L:12D and 18L:6D conditions. GS was present in Müller cells and GLAST expressed in Müller cell processes and photoreceptor inner segments. GLAST expression was decreased in 24L:0D condition, and the expression levels between 12L:12D and 18L:6D, though increased marginally, were statistically insignificant. Similar was the case with GS expression that significantly decreased in 24L:0D condition. Our previous study with chicks exposed to 2000 lux reported increased retinal glutamate level in 24L:0D condition. The present results indicate that constant light induces decreased expressions of GLAST and GS, a condition that might aggravate glutamate-mediated neurotoxicity and delay neuroprotection in a cone-dominated retina.

Differential Expression of AQP1 and AQP4 in Avascular Chick Retina Exposed to Moderate Light of Variable Photoperiods.

Aquaporins (AQPs) are integral membrane proteins which maintain cellular water and ion homeostasis. Alterations in AQP expression have been reported in rod-dominated rodent retinas exposed to light. In rodents and also in birds, light of moderate intensities (700-2000 lux) damages the retina, though detailed changes were not examined in birds. The aim of our study was to see if light affects cone dominated retinas, which would be reflected in expression levels of AQPs. We examined AQP1 and AQP4 expressions in chick retina exposed to 2000 lux under 12 h light:12 h dark (12L:12D; normal photoperiod), 18L:6D (prolonged photoperiod) and 24L:0D (constant light). Additionally, morphological changes, apoptosis (by TUNEL) and levels of glutamate and GFAP (a marker of injury) in the retina were examined to correlate these with AQP expressions. Constant light caused damage in outer and inner nuclear layer (ONL, INL) and ganglion cell layer (GCL). Also, there were associated increases in GFAP and glutamate levels in retinal extracts. In normal photoperiod, AQP1 was expressed in GCL, outer part of INL and photoreceptor inner segments of. AQP4 was additionally expressed in nerve fiber layer. Immunohistochemistry and Western blotting revealed over all decreased AQP1 and AQP4 expression in constant light condition compared to those in other two groups. The elevated GFAP and glutamate levels might be involved in the reduction of AQPs in constant light group. Such decreases in AQP expressions are perhaps linked with retinal cell damage seen in constant light condition, while their relatively enhanced expression in two other conditions may help in maintaining a normal retinal architecture, indicating their neuroprotective potential.

Retinal neuroprotective effects of quercetin in streptozotocin-induced diabetic rats.

The aim of the present study was to evaluate the effects of Quercetin (Qctn), a plant based flavonol, on retinal oxidative stress, neuroinflammation and apoptosis in streptozotocin-induced diabetic rats. Qctn treatment (25- and 50 mg/kg body weight) was given orally for six months in diabetic rats. Retinal glutathione (GSH) and antioxidant enzymes [superoxide dismutase (SOD) and catalase (CAT)] were estimated using commercially available assays, and inflammatory cytokines levels [tumor necrosis factor-α (TNF-α), Interleukin-1ß (IL-1ß)] were estimated by ELISA method. Immunofluorescence and western blot studies were performed for nuclear factor kappa B (NF-kB), caspase-3, glial fibrillary acidic protein (GFAP) and aquaporin-4 (AQP4) expressions. Structural changes were evaluated by light microscopy. In the present study, retinal GSH levels and antioxidant enzyme (SOD and CAT) activities were significantly decreased in diabetic group as compared to normal group. However, in Qctn-treated rats, retinal GSH levels were restored close to normal levels and positive modulation of antioxidant enzyme activities was observed. Diabetic retinas showed significantly increased expression of pro-inflammatory cytokines (TNF-α and IL-1ß) as compared to that in normal retinas, while Qctn-treated retinas showed significantly lower levels of cytokines as compared to diabetic retinas. Light microscopy showed significantly increased number of ganglion cell death and decreased retinal thickness in diabetic group compared to those in normal retina; however, protective effect of Qctn was seen. Increased apoptosis in diabetic retina is proposed to be mediated by overexpression of NF-kB and caspase-3. However, Qctn showed inhibitory effects on NF-kB and caspase-3 expression. Microglia showed upregulated GFAP expression, and inflammation of Müller cells resulted in edema in their endfeet and around perivascular space in nerve fiber layer in diabetic retina, as observed through AQP4 expression. However, Qctn treatments inhibited diabetes-induced increases in GFAP and AQP4 expression. Based on these findings, it can be concluded that bioflavonoids, such as Qctn can be effective for protection of diabetes induced retinal neurodegeneration and oxidative stress.

Simultaneous estimation of rutin and donepezil through RP-HPLC: implication in pharmaceutical and biological samples.

Aim: A HPLC method was developed and validated for the novel combination of rutin (RN) and donepezil (DNP). Materials & methods: RN and DNP were simultaneously eluted through a C18 column (Ø 150 × 4.6 mm) with a 60:40 v/v ratio of 0.1% formic acid aqueous solution to methanol at 0.5 ml/min. Results: The purposed method was found linear, selective, reproducible, accurate and precise with percent RSD less than 2. The limit of quantification for RN and DNP was found 3.66 and 3.25 µg/ml, respectively. Conclusion: Validated as per the ICH guidelines, the developed method efficiently quantified RN and DNP co-loaded in DQAsomes (121 nm) estimating matrix effect, release profile, entrapment efficiency, loading efficiency and in vivo plasma kinetics.

[Box: see text].

Hesperetin rescues retinal oxidative stress, neuroinflammation and apoptosis in diabetic rats.

The purpose of the study was to evaluate the effects of hesperetin (Hsp) on diabetes-induced retinal oxidative stress, neuroinflammation and apoptosis in rats. The Hsp treatment (100 mg/kg body weight) was carried for twenty four weeks in STZ-induced diabetic rats and evaluated for antioxidant (Superoxide dismutase; SOD, Catalase; CAT and glutathione; GSH) enzymes, inflammatory cytokines (TNF-α, IL-1ß), caspase-3, glial fibrillary acidic protein (GFAP) and aquaporin-4(AQP4) expression. Histological changes were evaluated by light and transmission electron microscopic (LM and TEM) studies. Retinal GSH levels and anti-oxidant enzymes (SOD and CAT) activity were significantly decreased in diabetic group as compared to normal group. However, in Hsp-treated rats, retinal GSH levels were restored close to normal levels and positive modulation of anti-oxidant enzyme activity was observed. Diabetic retinae showed significantly increased expression of Pro-inflammatory cytokines (TNF-α and IL-1ß) as compared to normal retinae. While Hsp-treated retinae showed significantly lower levels of cytokines as compared to diabetic retinae. Diabetic retinae showed increased caspase-3, GFAP and AQP4 expression. However, Hsp-treated retinae showed inhibitory effect on caspase-3, GFAP and AQP4 expression. LM images showed edematous Müller cell endfeet, and also degenerated photoreceptor layer; however, protective effect of Hsp was seen on Müller cell processes and photoreceptors. TEM study showed increased basement membrane (BM) thickness in diabetic retina, while relatively thin BM was recorded in Hsp-treated retina. It can be postulated that dietary flavanoids, like Hsp, can be effective for the prevention of diabetes induced neurovascular complications such as diabetic retinopathy.

Technology Transfer of a Validated RP-HPLC Method for the Simultaneous Estimation of Andrographolide and Paclitaxel in Application to Pharmaceutical Nanoformulation.

Many analytical methods are reported for simultaneous estimation of pharmaceutical dosages form. However, only a few are reproducible at an industrial scale. The proposed research aims to establish a technology transfer (TT) protocol between two laboratories (Lab-X, originator) with binary and (Lab-Y, receiver) with quaternary high-performance liquid chromatography (HPLC) system. Thus, utilizing reverse-phase HPLC (RP-HPLC), a robust, sensitive and repeatable analytical method has been developed, validated and TT between two laboratories for simultaneous estimation of Andrographolide (AG) and Paclitaxel (PTX). The method has been developed on a Phenomenex Luna C18 column (150 x 4.6, 5) sustained at 40°C and validated under the International Conference on Harmonisation (ICH) Q2 (R1) regulatory guideline and TT USP chapter 1224. The mobile phase consisted of MilliQ (pH = 3) and a combination of acetonitrile and methanol (1:1) in the ratio 50:50 with a flow rate of 0.45 mL/min, linear gradient elution in both labs. The AG and PTX were detected on the PDA detector at 224 and 227 nm wavelength with retention time of 4.5 ± 0.34 and 8.2 ± 0.02 min and limit of detection was found 0.028 ± 0.004 µg/mL, and 0.028 ± 0.0007 µg/mL, whereas limit of quantification as 0.086 ± 0.011 µg/mL and 0.088 ± 0.0014 µg/mL respectively in both labs. Throughout, this approach we have proved that proposed method is repeatable in both labs, and it can be used to quantify AG and PTX in developed pharmaceutical nano-formulations.

Tauroursodeoxycholic Acid Alleviates Endoplasmic Reticulum Stress-Mediated Visual Deficits in Diabetic tie2-TNF Transgenic Mice via TGR5 Signaling.

Purpose: This study evaluated if tauroursodeoxycholic acid (TUDCA) alleviates pro-inflammatory and endoplasmic reticulum (ER) stress-mediated visual deficits in diabetic tie2-TNF transgenic mice via Takeda G protein-coupled receptor 5 (TGR5) receptor signaling. Methods: Adult tie2-TNF transgenic or age-matched C57BL/6J (wildtype, WT) mice were made diabetic and treated subcutaneously with TUDCA. After 4 weeks, visual function, vascular permeability, immunohistology, and molecular analyses were assessed. Human retinal endothelial cells (HRECs) silenced for TGR5, followed by TNF and high glucose (HG) stress-mediated endothelial permeability, and transendothelial migration of activated leukocytes were assessed with TUDCA in vitro. Results: Compared with WT mice, tie2-TNF mice showed a decreased visual function correlated with a decrease in protein kinase C α (PKCα) in rod bipolar cells, and increased vascular permeability was further exacerbated in diabetic-tie2-TNF mice. Conversely, TUDCA alleviated these changes in diabetic mice. An increase in inflammation and ER stress in retina coincided with an increase in TGR5 expression in diabetic tie2-TNF mice that decreased with TUDCA. In vitro, HRECs exposed to TNF+HG demonstrated >2-fold increase in TGR5 expression, a 3-fold increase in leukocyte transmigration with a concomitant increase in permeability. Although TUDCA reversed these effects, HRECs silenced for TGR5 and challenged with TUDCA or TGR5 agonist failed to reverse the TNF+HG induced effects. Conclusions: Our data suggest that TUDCA will serve as an excellent therapeutic agent for diabetic complications addressing both vascular and neurodegenerative changes in the retina. Perturbation of the TGR5 receptor in the retina might play a role in linking retinal ER stress to neurovascular dysfunction in diabetic retinopathy.

Communication in non-communicable diseases (NCDs) and role of immunomodulatory nutraceuticals in their management.

Conveyance of pathogens between organisms causes communicable diseases. On the other hand, a non-communicable disease (NCD) was always thought to have no causative transmissible infective agents. Today, this clear distinction is increasingly getting blurred and NCDs are found to be associated with some transmissible components. The human microbiota carries a congregation of microbes, the majority and the most widely studied being bacteria in the gut. The adult human gut harbors ginormous inhabitant microbes, and the microbiome accommodates 150-fold more genes than the host genome. Microbial communities share a mutually beneficial relationship with the host, especially with respect to host physiology including digestion, immune responses, and metabolism. This review delineates the connection between environmental factors such as infections leading to gut dysbiosis and NCDs and explores the evidence regarding possible causal link between them. We also discuss the evidence regarding the value of appropriate therapeutic immunomodulatory nutritional interventions to reduce the development of such diseases. We behold such immunomodulatory effects have the potential to influence in various NCDs and restore homeostasis. We believe that the beginning of the era of microbiota-oriented personalized treatment modalities is not far away.

Appraisal of fluoroquinolone-loaded carubinose-linked hybrid nanoparticles for glycotargeting to alveolar macrophages.

There is a curious case in Alveolar macrophages (AM), the frontline defence recruits that contain the spread of all intruding bacteria. In response to Mycobacterium tuberculosis (M.tb), AM either contain the spread or are modulated by M.tb to create a region for their replication. The M.tb containing granulomas so formed are organised structures with confined boundaries. The limited availability of drugs inside AM aid drug tolerance and poor therapeutic outcomes in diseases like tuberculosis. The present work proves the glycotargeting efficiency of levofloxacin (LVF) to AM. The optimised formulation developed displayed good safety with 2% hemolysis and a viability of 61.14% on J774A.1 cells. The physicochemical characterisations such as Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) proved that carubinose linkage was accomplished and LVF is entrapped inside carubinose-linked hybrid formulation (CHF) and hybrid formulation (HF) in amorphous form. The transmission electron microscopy (TEM) images revealed a core-shell structure of HF. The particle size of 471.5 nm estimated through dynamic light scattering (DLS) is enough to achieve active and passive targeting to AM. The nanoparticle tracking analysis (NTA) data revealed that the diluted samples were free from aggregates. Fluorescence-activated cell sorting (FACS) data exhibited excellent uptake via CHF (15 times) and HF(3 times) with reference to plain fluorescein isothiocyanate (FITC). The pharmacokinetic studies revealed that CHF and HF release the entrapped moiety LVF in a controlled manner over 72 h. The stability studies indicated that the modified formulation remains stable over 6 months at 5 ± 3℃. Hence, hybrid systems can be efficiently modified via carubinose to target AM via the parenteral route.

Effective uptake of folate-functionalized ethionamide-loaded hybrid system: targeting alveolar macrophages.

Aim: To assess the targeting ability of hybrid nanosystems functionalized with folate. It also aimed to reduce stomach intolerance by substituting the oral route for parenteral delivery. Method: The nanosystems, prepared by nanoprecipitation technique, utilized a one-step method to prepare nanoparticles followed by surface functionalization through adsorption. The prepared nanosystems underwent physical characterization, in vitro and in vivo evaluations. Result: The nanosystems were effective in targeting the alveolar macrophages. Ethionamide was released from the formulation over 5 days. Fourier-transform infrared results proved the structural characteristics, and the positive charge further improved the targeting efficacy on the functionalized system. Conclusion: The hybrid formulation improved the release characteristics. Reduction in dosing frequency due to prolonged release improves compliance with the dosage regimen.

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.

Visual deficits after traumatic brain injury.

Traumatic brain injury (TBI) is frequently described as any head injury ceasing the brain's normal function. Anatomically, developmentally, and physiologically, the eye is deemed as an extension of the brain. Vision in TBI is underrepresented, and the number of active clinical trials in this field are sparse. Frequently, visual problems are overlooked at the time of TBI, often resulting in progressive vision loss, lengthening, and impairing rehabilitation. TBI can be either penetrative or non-penetrative, associated with degeneration of neurons, apoptotic cell death, inflammation, microglial activation, hemorrhage associated with vascular dysfunction; however, precise animal modeling that mimics the extensive visual deficits of TBI pathology remain elusive. Recent works in both the diagnostics and therapeutics fields are starting to make substantial progress in the right direction. Discussion of current advancements in TBI animal models and the recent pathophysiological findings related to the neuro-glia-vascular unit (NVU) will help elucidate novel targets for potential therapeutics lines. Only over the past decade have newer pharmaceutical and stem cell-based treatments begun to come to light. The potency for these new lines of TBI specific curatives will be discussed along with the review of current blast-induced TBI models, providing potential directions for future research.

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.

Changes in the Inner Retinal Cells after Intense and Constant Light Exposure in Sprague-Dawley Rats.

Light insult causes photoreceptor death. Few studies reported that continuous exposure to light affects horizontal, Müller and ganglion cells. We aimed to see the effect of constant light exposure on bipolar and amacrine cells. Adult Sprague-Dawley rats were exposed to 300 or 3000 lux for 7 days in 12-h light: 12-h dark cycles (12L:12D). The latter group was then exposed to 24L:0D for 48 h to induce significant damage. The same animals were reverted to 300 lux and reared for 15 days in 12L:12D cycles. They were sacrificed on different days to find the degree of retinal recovery, if any, from light injury. Besides photoreceptor death, continuous light for 48 h resulted in downregulation of parvalbumin in amacrine cells and recoverin in cone bipolar cells (CBC). Rod bipolar cells (RBC) maintained an unaltered pattern of PKC-α expression. Upon reversal, there were increased expressions of parvalbumin in amacrine cells and recoverin in CBC, while RBC showed an increasing trend of PKC-α expression. The data show that damage in bipolar and amacrine cells after exposure to intense, continuous light can be ameliorated upon reversal to normal LD cycles to which the animals were initially acclimated to.

GRP78 translocation to the cell surface and O-GlcNAcylation of VE-Cadherin contribute to ER stress-mediated endothelial permeability.

Increased O-GlcNAcylation, a well-known post-translational modification of proteins causally linked to various detrimental cellular functions in pathological conditions including diabetic retinopathy (DR). Previously we have shown that endothelial activation induced by inflammation and hyperglycemia results in the endoplasmic reticulum (ER) stress-mediated intercellular junction alterations accompanied by visual deficits in a tie2-TNF-α transgenic mouse model. In this study, we tested the hypothesis that increased ER stress via O-GlcNAcylation of VE-Cadherin likely contribute to endothelial permeability. We show that ER stress leads to GRP78 translocation to the plasma membrane, increased O-GlcNAcylation of proteins, particularly VE-Cadherin resulting in a defective complex partnering leading to the loss of retinal endothelial barrier integrity and increased transendothelial migration of monocytes. We further show an association of GRP78 with the VE-Cadherin under these conditions. Interestingly, cells exposed to ER stress inhibitor, tauroursodeoxycholic acid partially mitigated all these effects. Our findings suggest an essential role for ER stress and O-GlcNAcylation in altering the endothelial barrier function and reveal a potential therapeutic target in the treatment of DR.

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.

Adipose stem cells and their paracrine factors are therapeutic for early retinal complications of diabetes in the Ins2Akita mouse.

BACKGROUND: Early-stage diabetic retinopathy (DR) is characterized by neurovascular defects. In this study, we hypothesized that human adipose-derived stem cells (ASCs) positive for the pericyte marker CD140b, or their secreted paracrine factors, therapeutically rescue early-stage DR features in an Ins2Akita mouse model. METHODS: Ins2Akita mice at 24 weeks of age received intravitreal injections of CD140b-positive ASCs (1000 cells/1 µL) or 20× conditioned media from cytokine-primed ASCs (ASC-CM, 1 µL). Age-matched wildtype mice that received saline served as controls. Visual function experiments and histological analyses were performed 3 weeks post intravitreal injection. Biochemical and molecular analyses assessed the ASC-CM composition and its biological effects. RESULTS: Three weeks post-injection, Ins2Akita mice that received ASCs had ameliorated decreased b-wave amplitudes and vascular leakage but failed to improve visual acuity, whereas Ins2Akita mice that received ASC-CM demonstrated amelioration of all aforementioned visual deficits. The ASC-CM group demonstrated partial amelioration of retinal GFAP immunoreactivity and DR-related gene expression but the ASC group did not. While Ins2Akita mice that received ASCs exhibited occasional (1 in 8) hemorrhagic retinas, mice that received ASC-CM had no adverse complications. In vitro, ASC-CM protected against TNFα-induced retinal endothelial permeability as measured by transendothelial electrical resistance. Biochemical and molecular analyses demonstrated several anti-inflammatory proteins including TSG-6 being highly expressed in cytokine-primed ASC-CM. CONCLUSIONS: ASCs or their secreted factors mitigate retinal complications of diabetes in the Ins2Akita model. Further investigation is warranted to determine whether ASCs or their secreted factors are safe and effective therapeutic modalities long-term as current locally delivered therapies fail to effectively mitigate the progression of early-stage DR. Nonetheless, our study sheds new light on the therapeutic mechanisms of adult stem cells, with implications for assessing relative risks/benefits of experimental regenerative therapies for vision loss.