Lacrimal gland regeneration: The unmet challenges and promise for dry eye therapy.

Dry eye disease (DED) is a common multifactorial disease of the tear film and the ocular surface. The problem of DED has gained attention globally, with millions of people affected by the disorder. Although the treatment strategies for DED have significantly evolved over time, most of the existing modalities fall under the category of standard palliative care when viewed from a long-term perspective. To address these limitations, different approaches have been explored by various groups to uncover alternative treatment strategies that can contribute to a full regeneration of the damaged lacrimal gland, which is responsible for producing the major aqueous component of the tear film. For this, multiple groups have investigated the role of lacrimal gland cells in DED based on their regenerating, homing, and differentiating capabilities. In this review, we discuss in detail the therapeutic mechanisms and regenerative strategies that can potentially be applied for lacrimal gland regeneration as well as their therapeutic applications. This review mainly focuses on aqueous deficiency dry eye disease (ADDE) caused by lacrimal gland dysfunction and possible future treatment strategies. The current key findings from cell and tissue-based regenerative therapy modalities that could be utilised to achieve lacrimal gland tissue regeneration are summarized. In addition, this review summarises the available literature from in vitro to in vivo studies, their limitations in relation to lacrimal gland regeneration and the possible clinical applications. Finally, current issues and unmet needs of cell-based therapies in providing complete lacrimal gland tissue regeneration are discussed.

The effect of Rho Kinase inhibition on corneal nerve regeneration in vitro and in vivo.

PURPOSE: Impairment of corneal nerves can lead to neurotrophic keratopathy accompanied with severe ocular surface damage, which due to limited treatment options, can result in severe visual deterioration. This study evaluates a possible new treatment by enhancing the corneal nerve regeneration using a Rho Kinase inhibitor (Y27632). ROCK is known to play an important role in regulating cell morphology, adhesion and motility but little is known about its role in corneal nerve regeneration. METHODS: Effects of ROCK inhibition on murine peripheral nerves was assessed in single cell- and wound healing assays as well as a 3D in vitro model. Furthermore, Sholl analysis evaluating neuronal branching and life-death assays evaluating toxicity of the inhibitor were performed. An in vivo mouse model was established, with monitoring weekly corneal nerve regrowth using confocal microscopy. Additionally, corneal nerve fiber length was evaluated by immunofluorescence staining. Underlying pathways were examined by qrtPCR. RESULTS: ROCK inhibition leads to a significant enhancement of fiber growth in vitro. Sholl analysis revealed a higher degree of branching of treated fibers. Cytotoxicity assay showed no influence of Y27632 on cellular survival. In vivo measurement revealed significant enhanced regeneration after injury in the treated group. QrtPCR of trigeminal ganglia confirmed ROCK knock-down as well as altered pathways. CONCLUSION: The inhibition of ROCK after corneal nerve injury resulted in an enhanced regrowth of fibers in vitro and in vivo. This might be a step towards a new therapeutic concept for the treatment of impaired corneal nerves in diseases such as neurotrophic keratopathy.

Cell sheet technology: Influence of culture conditions on in vitro-cultivated corneal stromal tissue for regenerative therapies of the ocular surface.

The in vitro reconstruction of stromal tissue by long-term cultivation of corneal fibroblasts is a smart approach for regenerative therapies of ocular surface diseases. However, systematic investigations evaluating optimized cultivation protocols for the realization of a biomaterial are lacking. This study investigated the influence of supplements to the culture media of human corneal fibroblasts on the formation of a cell sheet consisting of cells and extracellular matrix. Among the supplements studied are vitamin C, fetal bovine serum, L-glutamine, components of collagen such as L-proline, L-4-hydroxyproline and glycine, and TGF-ß1, bFGF, IGF-2, PDGF-BB and insulin. After long-term cultivation, the proliferation, collagen and glycosaminoglycan content and light transmission of the cell sheets were examined. Biomechanical properties were investigated by tensile tests and the ultrastructure was characterized by electron microscopy, small-angle X-ray scattering, antibody staining and ELISA. The synthesis of extracellular matrix was significantly increased by cultivation with insulin or TGF-ß1, each with vitamin C. The sheets exhibited a high transparency and suitable material properties. The production of a transparent, scaffold-free, potentially autologous, in vitro-generated construct by culturing fibroblasts with extracellular matrix synthesis-stimulating supplements represents a promising approach for a biomaterial that can be used for ocular surface reconstruction in slowly progressing diseases.

Decellularized porcine conjunctiva as an alternative substrate for tissue-engineered epithelialized conjunctiva.

PURPOSE: The long-term success of visual rehabilitation in patients with severe conjunctival scarring is reliant on the reconstruction of the conjunctiva with a suitable substitute. The purpose of this study is the development and investigation of a re-epithelialized conjunctival substitute based on porcine decellularized conjunctiva (PDC). METHODS: PDC was re-epithelialized either with pre-expanded human conjunctival epithelial cells (PDC + HCEC) or with a human conjunctival explant placed directly on PDC (PDC + HCEx). Histology and immunohistochemistry were performed to evaluate epithelial thickness, proliferation (Ki67), apoptosis (Caspase 3), goblet cells (MUC5AC), and progenitor cells (CK15, ΔNp63, ABCG2). The superior construct (PDC + HCEx) was transplanted into a conjunctival defect of a rabbit (n = 6). Lissamine green staining verified the epithelialization in vivo. Orbital tissue was exenterated on day 10 and processed for histological and immunohistochemical analysis to examine the engrafted PDC + HCEx. A human-specific antibody was used to detect the transplanted cells. RESULTS: From day-14 in vitro onward, a significantly thicker epithelium and greater number of cells expressing Ki67, CK15, ΔNp63, and ABCG2 were noted for PDC + HCEx versus PDC + HCEC. MUC5AC-positive cells were found only in PDC + HCEx. The PDC + HCEx-grafted rabbit conjunctivas were lissamine-negative during the evaluation period, indicating epithelial integrity. Engrafted PDC + HCEx showed preserved progenitor cell properties and an increased number of goblet cells comparable to those of native conjunctiva. CONCLUSION: Placing and culturing a human conjunctival explant directly on PDC (PDC + HCEx) enables the generation of a stable, stratified, goblet cell-rich construct that could provide a promising alternative conjunctival substitute for patients with extensive conjunctival stem and goblet cell loss.

Decellularized human corneal stromal cell sheet as a novel matrix for ocular surface reconstruction.

The shortage of donor corneas as well as the limitations of tissue substitutes leads to the necessity to develop alternative materials for ocular surface reconstruction. Corneal surface substitutes must fulfill specific requirements such as high transparency, low immunogenicity, and mechanical stability combined with elasticity. This in vitro study evaluates a decellularized matrix secreted from human corneal fibroblasts (HCF) as an alternative material for ocular surface reconstruction. HCF from human donors were cultivated with the supplementation of vitamin C to form a stable and thick matrix. Furthermore, due to enhanced cultivation time, a three-dimensional like multilayered construct which partly mimics the complex structure of the corneal stroma could be generated. The formed human cell-based matrices (so-called cell sheets [CS]) were subsequently decellularized. The complete cell removal, collagen content, ultrastructure, and cell toxicity of the decellularized CS (DCS) as well as biomechanical properties were analyzed. Surgical feasibility was tested on enucleated porcine eyes. After decellularization and sterilization, a transparent, thick, cell free, and sterile tissue substitute resulted, which allowed expansion of limbal epithelial stem cells with no signs of cytotoxicity, and good surgical feasibility. DCS seem to be a promising new corneal tissue substitute derived from human cells without the limitation of donor material; however, future in vivo studies are necessary to further elucidate its potential for ocular surface reconstruction.

Treatment of diabetic retinopathy through neuropeptide Y-mediated enhancement of neurovascular microenvironment.

Diabetic retinopathy (DR) is one of the most severe clinical manifestations of diabetes mellitus and a major cause of blindness. DR is principally a microvascular disease, although the pathogenesis also involves metabolic reactive intermediates which induce neuronal and glial activation resulting in disruption of the neurovascular unit and regulation of the microvasculature. However, the impact of neural/glial activation in DR remains controversial, notwithstanding our understanding as to when neural/glial activation occurs in the course of disease. The objective of this study was to determine a potential protective role of neuropeptide Y (NPY) using an established model of DR permissive to N-methyl-D-aspartate (NMDA)-induced excitotoxic apoptosis of retinal ganglion cells (RGC) and vascular endothelial growth factor (VEGF)-induced vascular leakage. In vitro evaluation using primary retinal endothelial cells demonstrates that NPY promotes vascular integrity, demonstrated by maintained tight junction protein expression and reduced permeability in response to VEGF treatment. Furthermore, ex vivo assessment of retinal tissue explants shows that NPY can protect RGC from excitotoxic-induced apoptosis. In vivo clinical imaging and ex vivo tissue analysis in the diabetic model permitted assessment of NPY treatment in relation to neural and endothelial changes. The neuroprotective effects of NPY were confirmed by attenuating NMDA-induced retinal neural apoptosis and able to maintain inner retinal vascular integrity. These findings could have important clinical implications and offer novel therapeutic approaches for the treatment in the early stages of DR.

MSC Transplantation Improves Lacrimal Gland Regeneration after Surgically Induced Dry Eye Disease in Mice.

Dry eye disease (DED) is a multifactorial disease characterized by a disrupted tear film homeostasis and inflammation leading to visual impairments and pain in patients. Aqueous-deficient dry eye (ADDE) causes the most severe progressions and depends mainly on the loss of functional lacrimal gland (LG) tissue. Despite a high prevalence, therapies remain palliative. Therefore, it is of great interest to develop new approaches to curatively treat ADDE. Mesenchymal stem/stromal cells (MSC) have been shown to induce tissue regeneration and cease inflammation. Moreover, an increasing amount of MSC was found in the regenerating LG of mice. Therefore, this study investigated the therapeutic effect of MSC transplantation on damaged LGs using duct ligation induced ADDE in mice. Due to the transplantation of sex-mismatched and eGFP-expressing MSC, MSC could be identified and detected until day 21. MSC transplantation significantly improved LG regeneration, as the amount of vital acinar structures was significantly increased above the intrinsic regeneration capacity of control. Additionally, MSC transplantation modulated the immune reaction as macrophage infiltration was delayed and TNFα expression decreased, accompanied by an increased IL-6 expression. Thus, the application of MSC appears to be a promising therapeutic approach to induce LG regeneration in patients suffering from severe DED/ADDE.

Analysis of lacrimal gland derived mesenchymal stem cell secretome and its impact on epithelial cell survival.

In situ regeneration of lacrimal gland (LG) tissue would be a promising approach to curatively treat dry eye disease (DED). Mesenchymal stem cells (MSC) exhibit therapeutic effects in a variety of pathological conditions and our group recently reported that their number increases in regenerating mouse LG. Since the therapeutic effects are suggested to arise from secreted trophic factors, the application of MSC-secreted proteins seems to be a promising approach to induce/enhance LG regeneration. Therefore, this study aims to optimize the isolation of murine LG-MSC and analyze their secretome to investigate its potential for LG epithelial cell survival in vitro. For optimization, LG-MSC were isolated by an explant technique or cell sorting and their secretome was investigated under normal and inflammatory conditions. Results showed that the secretome of MSC had beneficial effects on the viability of ethanol-damaged LG epithelial cells. Additional, Lipocalin-2, prosaposin, ras GTPase-activating protein-binding protein 1 (Rac1) and signal transducer and activator of transcription 1 (STAT1), proteins that were up-regulated under inflammatory conditions, further improved the cell survival of ethanol-damaged LG epithelial cells. Interestingly, recovery of cell viability was highest, when the cells were incubated with STAT1. Summarizing, this study identified promising proteins for further studies on LG regeneration.

Restoring retinal neurovascular health via substance P.

Regulation of vascular permeability plays a major role in the pathophysiology of visually threatening conditions such as retinal vein occlusion and diabetic retinopathy. Principally, several factors such as vascular endothelial growth factor (VEGF), are up-regulated or induced in response to hypoxia thus adversely affecting the blood-retinal barrier (BRB), resulting in retinal edema and neovascularisation. Furthermore, current evidence supports a dysregulation of the inner retinal neural-vascular integrity as a critical factor driving retinal ganglion cell (RGC) death and visual loss. The principal objective of this study was to interrogate whether Substance P (SP), a constitutive neurotransmitter of amacrine and ganglion cells, may protect against N-methyl-d-aspartate (NMDA)-induced excitotoxic apoptosis of ganglion cells and VEGF-induced vessel leakage in the retina. Tight junctional protein expression and a Vascular Permeability Image Assay were used to determine vascular integrity in vitro. The protective effect of SP on RGC was established in ex vivo retinal explants and in vivo murine models. After NMDA administration, a reduction in TUNEL+ cells and a maintained number of Brn-3a+ cells were found, indicating an inhibition of RGC apoptosis mediated by SP. Additionally, SP maintained endothelial tight junctions and decreased VEGF-induced vascular permeability. In conclusion, administration of SP protects against NMDA apoptosis of RGC and VEGF-induced endothelial barrier breakdown.

Müller Cells Stabilize Microvasculature through Hypoxic Preconditioning.

BACKGROUND/AIMS: Hypoxia of the retina is a common pathogenic drive leading to vision loss as a result of tissue ischemia, increased vascular permeability and ultimately retinal neovascularisation. Here we tested the hypothesis that Müller cells stabilize the neurovascular unit, microvasculature by suppression of HIF-1α activation as a result of hypoxic preconditioning. METHODS: Tube Formation Assay and In vitro Vascular Permeability Image Assay were used to analyze angiogenesis and vascular integrity. Seahorse XF Cell Mito Stress Test was used to measure mitochondrial respiration. Gene and protein expression were examined by qRTPCR, ELISA and western blot. RESULTS: Hypoxic insult induces a significant induction of proangiogenic factors including vascular endothelial growth factor (VEGF) and angiopoietinlike 4 (ANGPTL-4) resulting in angiogenesis and increased vascular permeability of vascular endothelial cells. Hypoxic preconditioning of a human retinal Müller glia cell line significantly attenuates HIF-1α activation through the inhibition of mTOR and concomitant induction of aerobic glycolysis, stabilizing endothelial cells. CONCLUSION: Hypoxic preconditioning of Müller cells confers a robust protection to endothelial cells, through the suppression of HIF1α activation and its downstream regulation of VEGF and ANGPTL-4.

Evaluation of Plastic-Compressed Collagen for Conjunctival Repair in a Rabbit Model.

IMPACT STATEMENT: Conjunctival integrity is crucial for a healthy ocular surface and visual acuity. In severe cases of inflammatory surface disorders or after trauma, thermal or chemical burns as well as after ocular surgery, a surgical reconstruction using conjunctival substitutes is required. Due to limitations of currently used substitutes, such as the amniotic membrane, there is a need for the development of new scaffolds of consistent quality for conjunctival reconstruction. This study explored the biocompatibility and surgical usability of plastic-compressed collagen as an alternative conjunctival substitute in a rabbit model.

Comparative analysis on the dynamic of lacrimal gland damage and regeneration after Interleukin-1α or duct ligation induced dry eye disease in mice.

The loss of functional lacrimal gland (LG) tissue causes quantitative tear deficiency and is the most common reason for the development of severe dry eye disease (DED). The induction of LG regeneration in situ would be a promising approach to curatively treat DED, but underlying mechanisms are mainly unclear. Therefore, this study aims to comparatively evaluate the dynamic of LG damage and regeneration in two mouse models in order to study mechanisms of LG regeneration. Male C57BL/6 J mice were used to induce damage to the right extraorbital LG either by a single interleukin (IL) 1α injection or a ligation of the secretory duct for 7 days. Fluorescein staining (FL) and LG wet weight were assessed. In addition, the dynamic of damage and regeneration of acini structures as well as inflammation and the appearance of progenitor cells were (immuno-) histologically evaluated on day 1, 2, 3, 5, 7 after IL-1α injection and day 3, 7, 14, 21, 28 after duct ligation (DL). While LG weight was only slightly affected after IL-1α injection, DL led to a significant decrease at day 7 followed by an increase after re-opening. Additionally, DL resulted in a more pronounced inflammatory reaction than IL-1α injection. After DL the infiltration with CD3+ T cells, CD138 + plasma cells and CD68 + macrophages increased, while IL-1α injection only caused an infiltration with CD68 + macrophages. Furthermore, the damage of LG structures was significantly higher after DL than after IL-1α injection. Accordingly, regeneration of LG was prolonged and only partial at day 28 after DL, whilst 5 days after IL-1α injection a complete LG completely regeneration was achieved. We also found a significantly increased number of nestin + mesenchymal stem cells in both models during injury phase. Our results showed that both models induce LG damage followed by a spontaneous regeneration of acini structures. IL-1α injection caused an immediate inflammation with a transient period of slight tissue damage. However, DL caused a more distinct tissue damage followed by a prolonged period of regeneration, which might make it appear more attractive to study regenerative therapies and their effects on LG regeneration.

snRPN controls the ability of neurons to regenerate axons.

BACKGROUND: Retinal ganglion cells (RGCs) of mammals lose the ability to regenerate injured axons during postnatal maturation, but little is known about the underlying molecular mechanisms. OBJECTIVE: It remains of particular importance to understand the mechanisms of axonal regeneration to develop new therapeutic approaches for nerve injuries. METHODS: Retinas from newborn to adult monkeys (Callithrix jacchus)1 were obtained immediately after death and cultured in vitro. Growths of axons were monitored using microscopy and time-lapse video cinematography. Immunohistochemistry, Western blotting, qRT-PCR, and genomics were performed to characterize molecules associated with axonal regeneration and growth. A genomic screen was performed by using retinal explants versus native and non-regenerative explants obtained from eye cadavers on the day of birth, and hybridizing the mRNA with cross-reacting cDNA on conventional human microarrays. Followed the genomic screen, siRNA experiments were conducted to identify the functional involvement of identified candidates. RESULTS: Neuron-specific human ribonucleoprotein N (snRPN) was found to be a potential regulator of impaired axonal regeneration during neuronal maturation in these animals. In particular, up-regulation of snRPN was observed during retinal maturation, coinciding with a decline in regenerative ability. Axon regeneration was reactivated in snRPN-knockout retinal ex vivo explants of adult monkey. CONCLUSION: These results suggest that coordinated snRPN-driven activities within the neuron-specific ribonucleoprotein complex regulate the regenerative ability of RGCs in primates, thereby highlighting a potential new role for snRPN within neurons and the possibility of novel postinjury therapies.

Generation and characterisation of decellularised human corneal limbus.

PURPOSE: Limbal epithelial stem cells (LESC) reside in a niche in the corneo-scleral transition zone. Deficiency leads to pain, corneal opacity, and eventually blindness. LESC transplantation of ex-vivo expanded human LESC on a carrier such as human amniotic membrane is a current treatment option. We evaluated decellularised human limbus (DHL) as a potential carrier matrix for the transplantation of LESC. METHODS: Human corneas were obtained from the local eye bank. The limbal tissue was decellularised by sodium desoxychelate and DNase solution and sterilised by γ-irradiation. Native limbus- and DHL-surface structures were assessed by scanning electron microscopy and collagen ultrastructure using transmission electron microscopy. Presence and preservation of limbal basement membrane proteins in native limbus and DHL were analysed immunohistochemically. Absence of DNA after decellularisation was assessed by Feulgen staining and DNA quantification. Presence of immune cells was explored by CD45 staining, and potential cytotoxicity was tested using a cell viability assay. RESULTS: In the DHL, the DNA content was reduced from 1.5 ± 0.3 µg/mg to 0.15 ± 0.01 µg/mg; the three-dimensional structure and the arrangement of the collagen fibrils were preserved. Main basement membrane proteins such as collagen IV, laminin, and fibronectin were still present after decellularisation and γ-irradiation. CD45-expressing cells were evident neither in the native limbus nor in the DHL. DHL did not convey cytotoxicity. CONCLUSIONS: The extracellular matrix (ECM) of the limbus provides a tissue specific morphology and three-dimensionality consisting of particular ECM proteins. It therefore represents a substantial component of the stem cell niche. The DHL provides a specific limbal niche surrounding, and might serve as an easily producible carrier matrix for LESC transplantation.

Decellularised conjunctiva for ocular surface reconstruction.

Conjunctival reconstruction is an integral component of ocular surface restoration. Decellularised tissues are frequently used clinically for tissue engineering. This study identifies porcine decellularised conjunctiva (PDC) and human decellularised conjunctiva (HDC) as promising substitutes for conjunctival reconstruction. PDC and HDC were nearly DNA-free, structurally intact and showed no cytotoxic effects in vitro, which was confirmed by DNA quantification, histology, transmission electron microscopy, collagen quantification and cytotoxicity assay. Comparing the biomechanical properties to amniotic membrane (AM), the most frequently applied matrix for ocular surface reconstruction today, the decellularised conjunctiva was more extensible and elastic but exhibited less tensile strength. The in vivo application in a rabbit model proofed significantly enhanced transplant stability and less suture losses comparing PDC and HDC to AM while none of the matrices induced considerable inflammation. Ten days after implantation, all PDC, 4 of 6 HDC but none of the AM transplants were completely integrated into the recipient conjunctiva with a partially multi-layered epithelium. Altogether, decellularised conjunctivas of porcine and human origin were superior to AM for conjunctival reconstruction after xenogeneic application in vivo. STATEMENT OF SIGNIFICANCE: Conjunctival integrity is essential for a healthy ocular surface and clear vision. Its reconstruction is required in case of immunological diseases, after trauma, chemical or thermal burns or surgery involving the conjunctiva. Due to limitations of currently used substitute tissues such as amniotic membrane, there is a need for the development of new matrices for conjunctival reconstruction. Decellularised tissues are frequently applied clinically for tissue engineering. The present study identifies porcine and human decellularised conjunctiva as biocompatible and well tolerated scaffolds with superior integration into the recipient conjunctiva compared to amniotic membrane. Decellularised conjunctiva depicts a promising substitute for conjunctival reconstruction in ophthalmology.

Evaluation of Decellularized Porcine Jejunum as a Matrix for Lacrimal Gland Reconstruction In Vitro for Treatment of Dry Eye Syndrome.

Purpose: Dry eye syndrome (DES) can cause blindness in severe cases, but mainly palliative treatments exist. A tissue-engineered lacrimal gland (LG) could provide a curative treatment. We aimed to evaluate decellularized porcine jejunum (SIS-Muc) as a scaffold for porcine LG epithelial cells. Methods: To evaluate SIS-Muc as a potential scaffold, basement membrane proteins in SIS-Muc and native LG were compared (immunohistochemistry [IHC]). Porcine LG epithelial cells cultured on plastic were characterized (immunocytochemistry), and their culture supernatant was compared with porcine tears (proteomics). Epithelial cells were then seeded onto SIS-Muc in either a static (cell crown) or dynamic culture (within a perfusion chamber) and metabolic (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and secretory capacities (ß-hexosaminidase assay), protein expression (IHC), and ultrastructure transmission electron microscopy (TEM) compared in each. Results: Collagen IV and laminin were found in both native LG and SIS-Muc. When cultured on plastic, LG epithelial cells expressed pan-cytokeratin, Rab3D, HexA, and produced mucins, but lysozyme and lactoferrin expression was nearly absent. Some porcine tear proteins (lipocalin-2 and lactoferrin) were found in LG epithelial cell culture supernatants. When LG cells were cultured on SIS-Muc, metabolic and ß-hexosaminidase activities were greater in dynamic cultures than static cultures (P < 0.05). In both static and dynamic cultures, cells expressed pan-cytokeratin, Rab3D, lysozyme, and lactoferrin and produced mucins, and TEM revealed cell polarization at the apical surface and cell-cell and cell-scaffold contacts. Conclusions: SIS-Muc is a suitable scaffold for LG cell expansion and may be useful toward reconstruction of LG tissue to provide a curative treatment for DES. Dynamic culture enhances cell metabolic and functional activities.

The interplay between histone deacetylases and rho kinases is important for cancer and neurodegeneration.

Rho associated coiled-coil containing kinases (ROCKs) respond to defined extra- and intracellular stimuli to control cell migration, cell proliferation, and apoptosis. Histone deacetylases (HDACs) are epigenetic modifiers that regulate nuclear and cytoplasmic signaling through the deacetylation of histones and non-histone proteins. ROCK and HDAC functions are important compounds of basic and applied research interests. Recent evidence suggests a physiologically important interplay between HDACs and ROCKs in various cells and organisms. Here we summarize the crosstalk between these enzymatic families and its implications for cancer and neurodegeneration.

Analysis of Histone Deacetylase-Dependent Effects on Cell Migration Using the Stripe Assay.

For normal embryonic development/morphogenesis, cell migration and homing are well-orchestrated and important events requiring specific cellular mechanisms. In diseases such as cancer deregulated cell migration represents a major problem. Therefore, numerous efforts are under way to understand the molecular mechanisms of tumor cell migration and to generate more efficient tumor therapies. Cell migration assays are one of the most commonly used functional assays. The wound-healing assay or the Boyden chamber assay are variations of these assays. Nearly all of them are two-dimensional assays and the cells can only migrate on one substrate at a time. This is in contrast to the in vivo situation where the cells are faced simultaneously with different surfaces and interact with different cell types. To approach this in vivo situation we used a modified version of the stripe assay designed by Bonhoeffer and colleagues to examine mechanisms of axonal guidance. The design of this assay allows cells to decide between two different substrates offered at the same time. Utilizing alternating neuronal substrates for migration analyses we can partially mimic the complex in vivo situation for brain tumor cells. Here we describe the detailed protocol to perform a modified version of the stripe assay in order to observe substrate-dependent migration effects in vitro, to analyze the effect of Rho-dependent kinases (ROCKS), of histone deacetylases (HDACs) and of other molecules on glioma cells.

Development of Causative Treatment Strategies for Lacrimal Gland Insufficiency by Tissue Engineering and Cell Therapy. Part 2: Reconstruction of Lacrimal Gland Tissue: What Has Been Achieved So Far and What Are the Remaining Challenges?

The lacrimal gland is located in the upper temporal compartment of the orbita, and along with the ocular surface, eye lids, and sensory and motor nerves forms the lacrimal functional unit (LFU). The LFU is responsible for producing, distributing, and maintaining the tear film in order to maintain a smooth, moist, and regular ocular surface epithelium such that appropriate refractive properties are achieved and the eyeball is protected against dust, debris, and pathogens. If the main lacrimal gland is impaired (due to either disease or injury), this balance is disrupted, and severe quantitative dry eye syndrome (DES) can develop. DES treatments remain palliative, with the most commonly used therapies being based on tear substitution, tear retention, and control of inflammation on the ocular surface. Causative treatments such as salivary gland transplantation have shown to reduce symptoms in very severe cases, however can cause problems on the ocular surface due to different properties of saliva and tears. Therefore, causative approaches for treating DES by regeneration or reconstruction of lacrimal gland tissue depending on disease severity seem highly appealing. This article reviews current approaches for in vitro reconstruction of lacrimal gland tissue. Finally, the limitations that must be overcome before a new, tissue-engineered therapy may be delivered to clinic will be discussed.