Effects of Micromachining on Anti-oxidant Elution from a Mechanically-Adaptive Polymer.

Intracortical microelectrodes (IMEs) can be used to restore motor and sensory function as a part of brain-computer interfaces in individuals with neuromusculoskeletal disorders. However, the neuroinflammatory response to IMEs can result in their premature failure, leading to reduced therapeutic efficacy. Mechanically-adaptive, resveratrol-eluting (MARE) neural probes target two mechanisms believed to contribute to the neuroinflammatory response by reducing the mechanical mismatch between the brain tissue and device, as well as locally delivering an antioxidant therapeutic. To create the mechanically-adaptive substrate, a dispersion, casting, and evaporation method is used, followed by a microfabrication process to integrate functional recording electrodes on the material. Resveratrol release experiments were completed to generate a resveratrol release profile and demonstrated that the MARE probes are capable of long-term controlled release. Additionally, our results showed that resveratrol can be degraded by laser-micromachining, an important consideration for future device fabrication. Finally, the electrodes were shown to have a suitable impedance for single-unit neural recording and could record single units in vivo.

Ultrasound-mediated drug-free theranostics for treatment of prostate cancer.

Lipid-shelled nanobubbles (NBs) can be visualized and activated using noninvasive ultrasound (US) stimulation, leading to significant bioeffects. Prior work demonstrates that active targeting of NBs to prostate-specific membrane antigen (PSMA) overexpressed in prostate cancer (PCa) results in enhanced cellular internalization and prolongs NB retention with persistent, cancer-cell specific acoustic activity. In this work, we hypothesized that tumor-accumulated PSMA-NBs combined with low frequency unfocused therapeutic US (TUS) will lead to selective damage and induce a specific therapeutic effect in PSMA-expressing tumors compared to PSMA-negative tumors. We observed that the internalized NBs and cellular compartments were disrupted after the PSMA-NB + TUS (targeted NB therapy or TNT) application, yet treated cells remained intact and viable. In vivo, PSMA-expressing tumors in mice receiving TNT treatment demonstrated a significantly greater extent of apoptosis (78.4 ± 9.3 %, p < 0.01) compared to controls. TNT treatment significantly inhibited the PSMA expressing tumor growth and increased median survival time by 103 %, p < 0.001). A significant reduction in tumor progression compared to untreated control was also seen in an orthotopic rabbit PCa model. Results demonstrate that cavitation of PSMA-NBs internalized via receptor-mediated endocytosis into target PCa cells using unfocused ultrasound results in significant, tumor-specific bioeffects. The effects, while not lethal to PSMA-expressing cancer cells in vitro, result in significant in vivo reduction in tumor progression in two models of PCa. While the mechanism of action of these effects is yet unclear, it is likely related to a locally-induced immune response, opening the door to future investigations in this area.

Assessing Tumor Microenvironment Characteristics and Stratifying EPR with a Nanobubble Companion Nanoparticle via Contrast-Enhanced Ultrasound Imaging.

The tumor microenvironment is characterized by dysfunctional endothelial cells, resulting in heightened vascular permeability. Many nanoparticle-based drug delivery systems attempt to use this enhanced permeability combined with impaired lymphatic drainage (a concept known as the 'enhanced permeability and retention effect' or EPR effect) as the primary strategy for drug delivery, but this has not proven to be as clinically effective as anticipated. The specific mechanisms behind the inconsistent clinical outcomes of nanotherapeutics have not been clearly articulated, and the field has been hampered by a lack of accessible tools to study EPR-associated phenomena in clinically relevant scenarios. While medical imaging has tremendous potential to contribute to this area, it has not been broadly explored. This work examines, for the first time, the use of multiparametric dynamic contrast-enhanced ultrasound (CEUS) with a novel nanoscale contrast agent to examine tumor microenvironment characteristics noninvasively and in real-time. We demonstrate that CEUS imaging can: (1) evaluate tumor microenvironment features and (2) be used to help predict the distribution of doxorubicin-loaded liposomes in the tumor parenchyma. CEUS using nanobubbles (NBs) was carried out in two tumor types of high (LS174T) and low (U87) vascular permeability, and time-intensity curve (TIC) parameters were evaluated in both models prior to injection of doxorubicin liposomes. Consistently, LS174T tumors showed significantly different TIC parameters, including area under the rising curve (2.7x), time to peak intensity (1.9x) and decorrelation time (DT, 1.9x) compared to U87 tumors. Importantly, the DT parameter successfully predicted tumoral nanoparticle distribution (r = 0.86 ± 0.13). Ultimately, substantial differences in NB-CEUS generated parameters between LS174T and U87 tumors suggest that this method may be useful in determining tumor vascular permeability and could be used as a biomarker for identifying tumor characteristics and predicting sensitivity to nanoparticle-based therapies. These findings could ultimately be applied to predicting treatment efficacy and to evaluating EPR in other diseases with pathologically permeable vasculature.

Ultrasound-mediated drug-free theranostics for treatment of prostate cancer.

Rationale: Lipid-shelled nanobubbles (NBs) can be visualized and activated using noninvasive ultrasound (US) stimulation, leading to significant bioeffects. We have previously shown that active targeting of NBs to prostate-specific membrane antigen (PSMA) overexpressed in prostate cancer (PCa) enhances the cellular internalization and prolongs retention of NBs with persistent acoustic activity (~hrs.). In this work, we hypothesized that tumor-accumulated PSMA-NBs combined with low frequency therapeutic US (TUS) will lead to selective damage and induce a therapeutic effect in PSMA-expressing tumors compared to PSMA-negative tumors. Methods: PSMA-targeted NBs were formulated by following our previously established protocol. Cellular internalization of fluorescent PSMA-NBs was evaluated by confocal imaging using late endosome/lysosome staining pre- and post-TUS application. Two animal models were used to assess the technique. Mice with dual tumors (PSMA expressing and PSMA negative) received PSMA-NB injection via the tail vein followed by TUS 1 hr. post injection (termed, targeted NB therapy or TNT). Twenty-four hours after treatment mice were euthanized and tumor cell apoptosis evaluated via TUNEL staining. Mice with single tumors (either PSMA + or -) were used for survival studies. Tumor size was measured for 80 days after four consecutive TNT treatments (every 3 days). To test the approach in a larger model, immunosuppressed rabbits with orthotopic human PSMA expressing tumors received PSMA-NB injection via the tail vein followed by TUS 30 min after injection. Tumor progression was assessed via US imaging and at the end point apoptosis was measured via TUNEL staining. Results: In vitro TNT studies using confocal microscopy showed that the internalized NBs and cellular compartments were disrupted after the TUS application, yet treated cells remained intact and viable. In vivo, PSMA-expressing tumors in mice receiving TNT treatment demonstrated a significantly greater extent of apoptosis (78.45 ± 9.3%, p < 0.01) compared to the other groups. TNT treatment significantly inhibited the PSMA (+) tumor growth and overall survival significantly improved (median survival time increase by 103%, p < 0.001). A significant reduction in tumor progression compared to untreated control was also seen in the rabbit model in intraprostatic (90%) and in extraprostatic lesions (94%) (p = 0.069 and 0.003, respectively). Conclusion: We demonstrate for the first time the effect of PSMA-targeted nanobubble intracellular cavitation on cancer cell viability and tumor progression in two animal models. Data demonstrate that the targeted nanobubble therapy (TNT) approach relies primarily on mechanical disruption of intracellular vesicles and the resulting bioeffects appear to be more specific to target cancer cells expressing the PSMA receptor. The effect, while not lethal in vitro, resulted in significant tumor apoptosis in vivo in both a mouse and a rabbit model of PCa. While the mechanism of action of these effects is yet unclear, it is likely related to a locally-induced immune response, opening the door to future investigations in this area.

Wnt/ß-Catenin Signaling Pathway Is Necessary for the Specification but Not the Maintenance of the Mouse Retinal Pigment Epithelium.

ß-Catenin (Ctnnb1) has been shown to play critical roles in the development and maintenance of epithelial cells, including the retinal pigment epithelium (RPE). Ctnnb1 is not only a component of intercellular junctions in the epithelium, it also functions as a transcriptional regulator in the Wnt signaling pathway. To identify which of its functional modalities is critically involved in mouse RPE development and maintenance, we varied Ctnnb1 gene content and activity in mouse RPE lineage cells and tested their impacts on mouse eye development. We found that a Ctnnb1 double mutant (Ctnnb1dm), which exhibits impaired transcriptional activity, could not replace Ctnnb1 in the RPE, whereas Ctnnb1Y654E, which has reduced affinity for the junctions, could do so. Expression of the constitutively active Ctnnb1∆ex3 mutant also suppressed the development of RPE, instead facilitating a ciliary cell fate. However, the post-mitotic or mature RPE was insensitive to the loss, inactivation, or constitutive activation of Ctnnb1. Collectively, our results suggest that Ctnnb1 should be maintained within an optimal range to specify RPE through transcriptional regulation of Wnt target genes in the optic neuroepithelium.

Fabrication Methods and Chronic In Vivo Validation of Mechanically Adaptive Microfluidic Intracortical Devices.

Intracortical neural probes are both a powerful tool in basic neuroscience studies of brain function and a critical component of brain computer interfaces (BCIs) designed to restore function to paralyzed patients. Intracortical neural probes can be used both to detect neural activity at single unit resolution and to stimulate small populations of neurons with high resolution. Unfortunately, intracortical neural probes tend to fail at chronic timepoints in large part due to the neuroinflammatory response that follows implantation and persistent dwelling in the cortex. Many promising approaches are under development to circumvent the inflammatory response, including the development of less inflammatory materials/device designs and the delivery of antioxidant or anti-inflammatory therapies. Here, we report on our recent efforts to integrate the neuroprotective effects of both a dynamically softening polymer substrate designed to minimize tissue strain and localized drug delivery at the intracortical neural probe/tissue interface through the incorporation of microfluidic channels within the probe. The fabrication process and device design were both optimized with respect to the resulting device mechanical properties, stability, and microfluidic functionality. The optimized devices were successfully able to deliver an antioxidant solution throughout a six-week in vivo rat study. Histological data indicated that a multi-outlet design was most effective at reducing markers of inflammation. The ability to reduce inflammation through a combined approach of drug delivery and soft materials as a platform technology allows future studies to explore additional therapeutics to further enhance intracortical neural probes performance and longevity for clinical applications.

Effect of meteorological conditions on leisure walking: a time series analysis and the application of outdoor thermal comfort indexes.

Leisure walking is affected by meteorological conditions. However, it is still not clear what scales of meteorological conditions and thermal status affect the number of people who choose to leisure walk. Using a time series regression, this study examines the heat-leisure walking relationship by analyzing the effect of the seasons, weather, microclimate, and outdoor thermal comfort on walking count. Eight thermal indexes were selected to estimate the pedestrians' thermal comfort, and their predictive capacities in walking count were evaluated. Particular consideration was given to identifying heat thresholds of walking that determined the tolerance range of pedestrian heat stress. Four years of hourly daytime walking counts and publicly available ASOS meteorological data at Seoul-lo 7017, a pedestrian bridge in Seoul, were used for the analysis. Our findings indicate that walking count is correlated with seasonal climatic variations, with the highest number of pedestrians observed in fall and the lowest in summer. Moreover, air temperature played a vital role, showing that a 5.0 °C rise in temperature was associated with a 1.34% rise in the square root of the walking count. Its impact becomes greater when combined with intense solar radiation and higher absolute humidity. The heat threshold for walking was between 23.8 °C and 26.2 °C. Empirical model indexes showed the highest predictive capacity in walking count at approximately 30.0%, which was followed by rational model indexes at 28.0%.

Platelet-mimicking procoagulant nanoparticles augment hemostasis in animal models of bleeding.

Treatment of bleeding disorders using transfusion of donor-derived platelets faces logistical challenges due to their limited availability, high risk of contamination, and short (5 to 7 days) shelf life. These challenges could be potentially addressed by designing platelet mimetics that emulate the adhesion, aggregation, and procoagulant functions of platelets. To this end, we created liposome-based platelet-mimicking procoagulant nanoparticles (PPNs) that can expose the phospholipid phosphatidylserine on their surface in response to plasmin. First, we tested PPNs in vitro using human plasma and demonstrated plasmin-triggered exposure of phosphatidylserine and the resultant assembly of coagulation factors on the PPN surface. We also showed that this phosphatidylserine exposed on the PPN surface could restore and enhance thrombin generation and fibrin formation in human plasma depleted of platelets. In human plasma and whole blood in vitro, PPNs improved fibrin stability and clot robustness in a fibrinolytic environment. We then tested PPNs in vivo in a mouse model of thrombocytopenia where treatment with PPNs reduced blood loss in a manner comparable to treatment with syngeneic platelets. Furthermore, in rat and mouse models of traumatic hemorrhage, treatment with PPNs substantially reduced bleeding and improved survival. No sign of systemic or off-target thrombotic risks was observed in the animal studies. These findings demonstrate the potential of PPNs as a platelet surrogate that should be further investigated for the management of bleeding.

Investigation of the Feasibility of Ventricular Delivery of Resveratrol to the Microelectrode Tissue Interface.

(1) Background: Intracortical microelectrodes (IMEs) are essential to basic brain research and clinical brain-machine interfacing applications. However, the foreign body response to IMEs results in chronic inflammation and an increase in levels of reactive oxygen and nitrogen species (ROS/RNS). The current study builds on our previous work, by testing a new delivery method of a promising antioxidant as a means of extending intracortical microelectrodes performance. While resveratrol has shown efficacy in improving tissue response, chronic delivery has proven difficult because of its low solubility in water and low bioavailability due to extensive first pass metabolism. (2) Methods: Investigation of an intraventricular delivery of resveratrol in rats was performed herein to circumvent bioavailability hurdles of resveratrol delivery to the brain. (3) Results: Intraventricular delivery of resveratrol in rats delivered resveratrol to the electrode interface. However, intraventricular delivery did not have a significant impact on electrophysiological recordings over the six-week study. Histological findings indicated that rats receiving intraventricular delivery of resveratrol had a decrease of oxidative stress, yet other biomarkers of inflammation were found to be not significantly different from control groups. However, investigation of the bioavailability of resveratrol indicated a decrease in resveratrol accumulation in the brain with time coupled with inconsistent drug elution from the cannulas. Further inspection showed that there may be tissue or cellular debris clogging the cannulas, resulting in variable elution, which may have impacted the results of the study. (4) Conclusions: These results indicate that the intraventricular delivery approach described herein needs further optimization, or may not be well suited for this application.

mTORC1-induced retinal progenitor cell overproliferation leads to accelerated mitotic aging and degeneration of descendent Müller glia.

Retinal progenitor cells (RPCs) divide in limited numbers to generate the cells comprising vertebrate retina. The molecular mechanism that leads RPC to the division limit, however, remains elusive. Here, we find that the hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) in an RPC subset by deletion of tuberous sclerosis complex 1 (Tsc1) makes the RPCs arrive at the division limit precociously and produce Müller glia (MG) that degenerate from senescence-associated cell death. We further show the hyperproliferation of Tsc1-deficient RPCs and the degeneration of MG in the mouse retina disappear by concomitant deletion of hypoxia-induced factor 1-alpha (Hif1a), which induces glycolytic gene expression to support mTORC1-induced RPC proliferation. Collectively, our results suggest that, by having mTORC1 constitutively active, an RPC divides and exhausts mitotic capacity faster than neighboring RPCs, and thus produces retinal cells that degenerate with aging-related changes.

Retinoid Metabolism in the Degeneration of Pten-Deficient Mouse Retinal Pigment Epithelium.

In vertebrate eyes, the retinal pigment epithelium (RPE) provides structural and functional homeostasis to the retina. The RPE takes up retinol (ROL) to be dehydrogenated and isomerized to 11-cis-retinaldehyde (11-cis-RAL), which is a functional photopigment in mammalian photoreceptors. As excessive ROL is toxic, the RPE must also establish mechanisms to protect against ROL toxicity. Here, we found that the levels of retinol dehydrogenases (RDHs) are commonly decreased in phosphatase tensin homolog (Pten)-deficient mouse RPE, which degenerates due to elevated ROL and that can be rescued by feeding a ROL-free diet. We also identified that RDH gene expression is regulated by forkhead box O (FOXO) transcription factors, which are inactivated by hyperactive Akt in the Pten-deficient mouse RPE. Together, our findings suggest that a homeostatic pathway comprising PTEN, FOXO, and RDH can protect the RPE from ROL toxicity.

Tsg101 Is Necessary for the Establishment and Maintenance of Mouse Retinal Pigment Epithelial Cell Polarity.

The retinal pigment epithelium (RPE) forms a monolayer sheet separating the retina and choroid in vertebrate eyes. The polarized nature of RPE is maintained by distributing membrane proteins differentially along apico-basal axis. We found the distributions of these proteins differ in embryonic, post-natal, and mature mouse RPE, suggesting developmental regulation of protein trafficking. Thus, we deleted tumor susceptibility gene 101 (Tsg101), a key component of endosomal sorting complexes required for transport (ESCRT), in embryonic and mature RPE to determine whether ESCRT-mediated endocytic protein trafficking correlated with the establishment and maintenance of RPE polarity. Loss of Tsg101 severely disturbed the polarity of RPE, which forms irregular aggregates exhibiting non-polarized distribution of cell adhesion proteins and activation of epidermal growth factor receptor signaling. These findings suggest that ESCRT-mediated protein trafficking is essential for the development and maintenance of RPE cell polarity.

SLC6A20 transporter: a novel regulator of brain glycine homeostasis and NMDAR function.

Glycine transporters (GlyT1 and GlyT2) that regulate levels of brain glycine, an inhibitory neurotransmitter with co-agonist activity for NMDA receptors (NMDARs), have been considered to be important targets for the treatment of brain disorders with suppressed NMDAR function such as schizophrenia. However, it remains unclear whether other amino acid transporters expressed in the brain can also regulate brain glycine levels and NMDAR function. Here, we report that SLC6A20A, an amino acid transporter known to transport proline based on in vitro data but is understudied in the brain, regulates proline and glycine levels and NMDAR function in the mouse brain. SLC6A20A transcript and protein levels were abnormally increased in mice carrying a mutant PTEN protein lacking the C terminus through enhanced ß-catenin binding to the Slc6a20a gene. These mice displayed reduced extracellular levels of brain proline and glycine and decreased NMDAR currents. Elevating glycine levels back to normal ranges by antisense oligonucleotide-induced SLC6A20 knockdown, or the competitive GlyT1 antagonist sarcosine, normalized NMDAR currents and repetitive climbing behavior observed in these mice. Conversely, mice lacking SLC6A20A displayed increased extracellular glycine levels and NMDAR currents. Lastly, both mouse and human SLC6A20 proteins mediated proline and glycine transports, and SLC6A20 proteins could be detected in human neurons. These results suggest that SLC6A20 regulates proline and glycine homeostasis in the brain and that SLC6A20 inhibition has therapeutic potential for brain disorders involving NMDAR hypofunction.

Investigating the Association between Motor Function, Neuroinflammation, and Recording Metrics in the Performance of Intracortical Microelectrode Implanted in Motor Cortex.

Long-term reliability of intracortical microelectrodes remains a challenge for increased acceptance and deployment. There are conflicting reports comparing measurements associated with recording quality with postmortem histology, in attempts to better understand failure of intracortical microelectrodes (IMEs). Our group has recently introduced the assessment of motor behavior tasks as another metric to evaluate the effects of IME implantation. We hypothesized that adding the third dimension to our analysis, functional behavior testing, could provide substantial insight on the health of the tissue, success of surgery/implantation, and the long-term performance of the implanted device. Here we present our novel analysis scheme including: (1) the use of numerical formal concept analysis (nFCA) and (2) a regression analysis utilizing modern model/variable selection. The analyses found complimentary relationships between the variables. The histological variables for glial cell activation had associations between each other, as well as the neuronal density around the electrode interface. The neuronal density had associations to the electrophysiological recordings and some of the motor behavior metrics analyzed. The novel analyses presented herein describe a valuable tool that can be utilized to assess and understand relationships between diverse variables being investigated. These models can be applied to a wide range of ongoing investigations utilizing various devices and therapeutics.

Toward Standardization of Electrophysiology and Computational Tissue Strain in Rodent Intracortical Microelectrode Models.

Progress has been made in the field of neural interfacing using both mouse and rat models, yet standardization of these models' interchangeability has yet to be established. The mouse model allows for transgenic, optogenetic, and advanced imaging modalities which can be used to examine the biological impact and failure mechanisms associated with the neural implant itself. The ability to directly compare electrophysiological data between mouse and rat models is crucial for the development and assessment of neural interfaces. The most obvious difference in the two rodent models is size, which raises concern for the role of device-induced tissue strain. Strain exerted on brain tissue by implanted microelectrode arrays is hypothesized to affect long-term recording performance. Therefore, understanding any potential differences in tissue strain caused by differences in the implant to tissue size ratio is crucial for validating the interchangeability of rat and mouse models. Hence, this study is aimed at investigating the electrophysiological variances and predictive device-induced tissue strain. Rat and mouse electrophysiological recordings were collected from implanted animals for eight weeks. A finite element model was utilized to assess the tissue strain from implanted intracortical microelectrodes, taking into account the differences in the depth within the cortex, implantation depth, and electrode geometry between the two models. The rat model demonstrated a larger percentage of channels recording single unit activity and number of units recorded per channel at acute but not chronic time points, relative to the mouse model Additionally, the finite element models also revealed no predictive differences in tissue strain between the two rodent models. Collectively our results show that these two models are comparable after taking into consideration some recommendations to maintain uniform conditions for future studies where direct comparisons of electrophysiological and tissue strain data between the two animal models will be required.

Nano-Architectural Approaches for Improved Intracortical Interface Technologies.

Intracortical microelectrodes (IME) are neural devices that initially were designed to function as neuroscience tools to enable researchers to understand the nervous system. Over the years, technology that aids interfacing with the nervous system has allowed the ability to treat patients with a wide range of neurological injuries and diseases. Despite the substantial success that has been demonstrated using IME in neural interface applications, these implants eventually fail due to loss of quality recording signals. Recent strategies to improve interfacing with the nervous system have been inspired by methods that mimic the native tissue. This review focusses on one strategy in particular, nano-architecture, a term we introduce that encompasses the approach of roughening the surface of the implant. Various nano-architecture approaches have been hypothesized to improve the biocompatibility of IMEs, enhance the recording quality, and increase the longevity of the implant. This review will begin by introducing IME technology and discuss the challenges facing the clinical deployment of IME technology. The biological inspiration of nano-architecture approaches will be explained as well as leading fabrication methods used to create nano-architecture and their limitations. A review of the effects of nano-architecture surfaces on neural cells will be examined, depicting the various cellular responses to these modified surfaces in both in vitro and pre-clinical models. The proposed mechanism elucidating the ability of nano-architectures to influence cellular phenotype will be considered. Finally, the frontiers of next generation nano-architecture IMEs will be identified, with perspective given on the future impact of this interfacing approach.

Zinc increases the phagocytic capacity of canine peripheral blood phagocytes in vitro.

Zinc is a trace element that plays a central role in the immune system. In the present study, the effect of zinc on the phagocytic capacity of canine peripheral blood phagocytes was examined in vitro by flow cytometry. Zinc was used at a concentration of 100 microM, which preserved cell viability. Treatment with zinc did not directly affect the phagocytic capacity of peripheral blood polymorphonuclear neutrophils (PMN) and mononuclear cells (PBMC). However, it did directly enhance the phagocytic capacity of peripheral blood monocyte-rich cells. Moreover, the phagocytic capacity of PMN and monocyte-rich cells but not PBMC was remarkably enhanced by culture supernatants from PBMC but not PMN treated with zinc. Anti-recombinant canine (rc) tumor necrosis factor-alpha (TNF-alpha) polyclonal antibody (pAb) neutralized the enhancing effect of the culture supernatant from zinc-treated PBMC and this supernatant had higher TNF-alpha levels than the culture supernatant of untreated PBMC. Thus, zinc may stimulate canine PBMC to produce TNF-alpha, which enhances the phagocytic capacity of canine peripheral blood phagocytes.

Neuroprotective effects of human mesenchymal stem cells on dopaminergic neurons through anti-inflammatory action.

Parkinson's disease (PD) is a common, progressive neurodegenerative disorder caused by the loss of dopaminergic neurons in the substantia nigra (SN). Numerous studies have provided evidence suggesting that neuroinflammation plays an important role in the pathogenesis of PD. In this study, we used lipopolysaccharide (LPS)-induced in vitro and in vivo inflammation models to investigate whether human mesenchymal stem cells (hMSCs) have a protective effect on the dopaminergic system through anti-inflammatory mechanisms. The hMSC treatment significantly decreased LPS-induced microglial activation, tumor necrosis factor (TNF)-alpha, inducible nitric oxide synthase (iNOS) mRNA expression, and production of NO and TNF-alpha compared with the LPS-only treatment group. In co-cultures of microglia and mesencephalic dopaminergic neurons, hMSC treatment significantly decreased the loss of tyrosine hydroxylase-immunopositive (TH-ip) cells. The hMSC treatment in rats showed that TH-ip neuronal loss induced by LPS stimulation in the SN was considerably decreased and was clearly accompanied by a decrease in activation of microglia, as well as TNF-alpha and iNOS mRNA expression and production of TNF-alpha. These data suggest that hMSCs have a neuroprotective effect on dopaminergic neurons through anti-inflammatory actions mediated by the modulation of microglial activation. Along with various trophic effects and trans-differentiational potency, the anti-inflammatory properties of MSCs could have major therapeutic implications in the treatment of PD.