Evaluation of Corneal Cross-Linking for Treatment of Fungal Keratitis: Using Confocal Laser Scanning Microscopy on an Ex Vivo Human Corneal Model.

Purpose: Some previous reports have established the use of photoactivated chromophore-induced corneal cross-linking (PACK-CXL) in treating fungal keratitis. The results of these case reports have often been conflicting. To systematically study the effect of PACK-CXL in the management of Fusarium keratitis, we have developed an ex vivo model of human corneal infection using eye-banked human corneas. Methods: Sixteen healthy ex vivo human corneas were divided into four study groups: (1) untreated control, (2) cross-linked, (3) infected with fungal spores, and (4) infected with fungal spores and then cross-linked. All infected corneas were inoculated with Fusarium oxysporum spores. The PACK-CXL procedure was performed 24 hours post inoculation for group 4. For PACK-CXL treatment, the corneas were debrided of epithelium; then 1% (wt/vol) isotonic riboflavin was applied dropwise at 5-minute intervals for 30 minutes and during the course of UV-A cross-linking for another 30 minutes. The corneas were imaged using a confocal microscope at 48 hours post inoculation, and the Fusarium hyphal volume and spore concentration were calculated. Results: The infected and then cross-linked group had a significantly lower volume of Fusarium hyphae, compared to the infected (P = 0.001) group. In the infected and then cross-linked group there was significant inhibition of Fusarium sporulation compared with the infected (P = 0.007) group. Conclusions: A model of human corneal infection was successfully developed for investigation of the effects of PACK-CXL on fungal keratitis. A treatment regimen of combined UV-A/riboflavin-induced corneal cross-linking appears to be a valuable approach to inhibit the growth and sporulation of Fusarium and suppress the progression of fungal keratitis.

Regeneration of Corneal Epithelium With Dental Pulp Stem Cells Using a Contact Lens Delivery System.

PURPOSE: The corneal epithelium is sloughed off surface of the eye by the action of blinking and is continually replaced by division and maturation of the limbal stem cells (LSCs). In the case of injury or disease, LSCs can be lost or damaged to a point at which the corneal epithelial layer is no longer maintained. leading to LSC deficiencies (LSCDs). When this occurs, the opaque conjunctiva overgrows the anterior surface of the eye, leading to vision impairment or loss. Dental pulp stem cells (DPSCs) are promising candidates as autologous LSC substitutes. In this study, contact lenses (CLs) are used as a novel medical device to deliver DPSCs onto corneal surface to enhance corneal epithelium regeneration. METHODS: Dental pulp stem cells labeled with green fluorescent Qtracker 525 were seeded onto the pretreated CLs, allowed to adhere, then delivered to debrided human corneas. Expression of KRT3, 12, 13, and 19 was investigated by immunostaining, then standard and confocal microscopy. RESULTS: Dental pulp stem cells were successfully isolated, labeled, and delivered to the corneal surface using CLs. Following removal of CLs, confocal microscopy showed that the DPSCs had migrated onto the cornea. Coexpression of KRT12 and green fluorescent Qtracker 525 confirmed that the DPSCs had transdifferentiated into corneal epithelial progenitors. Delimitation of KRT 19 and green fluorescence provides evidence that Qtracker 525-labeled DPSCs establish a barrier to the invasion of the cornea by conjunctiva. CONCLUSIONS: In this study we show that DPSCs, delivered using CLs, can be used to enhance repair and regeneration of the human corneal epithelium.

Sex-biased inbreeding effects on reproductive success and home range size of the critically endangered black rhinoceros.

A central premise of conservation biology is that small populations suffer reduced viability through loss of genetic diversity and inbreeding. However, there is little evidence that variation in inbreeding impacts individual reproductive success within remnant populations of threatened taxa, largely due to problems associated with obtaining comprehensive pedigree information to estimate inbreeding. In the critically endangered black rhinoceros, a species that experienced severe demographic reductions, we used model selection to identify factors associated with variation in reproductive success (number of offspring). Factors examined as predictors of reproductive success were age, home range size, number of nearby mates, reserve location, and multilocus heterozygosity (a proxy for inbreeding). Multilocus heterozygosity predicted male reproductive success (p< 0.001, explained deviance >58%) and correlated with male home range size (p < 0.01, r(2) > 44%). Such effects were not apparent in females, where reproductive success was determined by age (p < 0.01, explained deviance 34%) as females raise calves alone and choose between, rather than compete for, mates. This first report of a 3-way association between an individual male's heterozygosity, reproductive output, and territory size in a large vertebrate is consistent with an asymmetry in the level of intrasexual competition and highlights the relevance of sex-biased inbreeding for the management of many conservation-priority species. Our results contrast with the idea that wild populations of threatened taxa may possess some inherent difference from most nonthreatened populations that necessitates the use of detailed pedigrees to study inbreeding effects. Despite substantial variance in male reproductive success, the increased fitness of more heterozygous males limits the loss of heterozygosity. Understanding how individual differences in genetic diversity mediate the outcome of intrasexual competition will be essential for effective management, particularly in enclosed populations, where individuals have restricted choice about home range location and where the reproductive impact of translocated animals will depend upon the background distribution in individual heterozygosity.

Isolation of adult stem cells and their differentiation to Schwann cells.

Peripheral nerve injuries are an economic burden for society in general and despite advanced microsurgical reconstruction of the damaged nerves the functional result is unsatisfactory with poor sensory recovery and reduced motor functions (Wiberg and Terenghi, Surg Technol Int 11:303-310, 2003). In the treatment of nerve injuries transplantation of a nerve graft is often necessary, especially in nerve gap injuries.Schwann cells (SC) are the key facilitators of peripheral nerve regeneration and are responsible for the formation and maintenance of the myelin sheath around axons in peripheral nerve fibers. They are essential for nerve regeneration after nerve injuries as they produce extracellular matrix molecules, integrins, and trophic factors providing guidance and trophic support for regenerating axons (Wiberg and Terenghi, Surg Technol Int 11:303-310, 2003; Bunge, J Neurol 242:S19-21, 1994; Ide, Neurosci Res 25:101-121, 1996; Mahanthappa et al. J Neurosci 16:4673-4683, 1996). However, the use of ex vivo cultured SC within conduits is limited in its clinical application because of the concomitant donor site morbidity and the slow growth of these cells in vitro (Tohill et al. Tissue Eng 10:1359-1367, 2004).Mesenchymal stem cells (MSC or bone marrow stromal cells) and adipose-derived stem cells (ASC) are easily accessible non-hematopoietic stem cells that have proven essential for research purposes due to their plasticity and ability to differentiate into several functional cell types. This alternative source of cells is relatively simple to isolate and expand in culture. We have demonstrated that MSC and ASC can trans-differentiate along a SC lineage with functional properties and growth factor synthesis activities similar to those of native SC and could provide nerve fiber support and guidance during nerve regeneration.

Morphological, molecular and functional differences of adult bone marrow- and adipose-derived stem cells isolated from rats of different ages.

Adult mesenchymal stem cells have self-renewal and multiple differentiation potentials, and play important roles in regenerative medicine. However, their use may be limited by senescence or age of the donor, leading to changes in stem cell functionality. We investigated morphological, molecular and functional differences between bone marrow-derived (MSC) and adipose-derived (ASC) stem cells isolated from neonatal, young and old rats compared to Schwann cells from the same animals. Immunocytochemistry, RT-PCR, proliferation assays, western blotting and transmission electron microscopy were used to investigate expression of senescence markers. Undifferentiated and differentiated ASC and MSC from animals of different ages expressed Notch-2 at similar levels; protein-38 and protein-53 were present in all groups of cells with a trend towards increased levels in cells from older animals compared to those from neonatal and young rats. Following co-culture with adult neuronal cells, dMSC and dASC from animals of all ages elicited robust neurite outgrowth. Mitotracker(®) staining was consistent with ultrastructural changes seen in the mitochondria of cells from old rats, indicative of senescence. In conclusion, this study showed that although the cells from aged animals expressed markers of senescence, aged MSC and ASC differentiated into SC-like cells still retain potential to support axon regeneration.

Phenotype of distinct primary sensory afferent subpopulations and caspase-3 expression following axotomy.

Specific sensory neuronal subpopulations show contrasting responses to peripheral nerve injury, as shown by the axotomy-induced death of many cutaneous sensory neurons whilst muscular sensory afferents survive an identical insult. We used a novel combination of retrograde neuronal tracing with immunohistochemistry and laser microdissection techniques, in order to describe the neurochemistry of medial gastrocnemius (muscular sensory afferents) and sural (cutaneous sensory afferents) branches of the rat sciatic nerve and relate this to the pro-apoptotic caspase-3 gene expression following nerve transection. Our results demonstrated distinctions in medial gastrocnemius and sural neuron populations with the most striking difference in the respective proportions of isolectin B4 (IB4) staining neurons (3.7 V 32.8%). The mean neuronal area of the medial gastrocnemius (MG) neurons was larger than that of the sural (SUR) neurons (1,070.8 V 646.2 µm²) and each phenotypic group was significantly smaller in sural neurons than in MG neurons. At 1 week post-axotomy, MG neurons markedly downregulated caspase-3, whilst SUR neurons upregulated caspase-3 gene expression; this may be attributable to the differing IB4-positive composition of the subpopulations. These findings provide further clarification in the understanding of two distinct neuronal populations used increasingly in nerve injury models.

Schwann-like adult stem cells derived from bone marrow and adipose tissue express γ-aminobutyric acid type B receptors.

γ-Aminobutyric acid type B receptors (GABA-B) are expressed in glial cells of the central and peripheral nervous systems, and recent evidence has shown their importance in modulating physiological parameters of Schwann cell (SC). SC play essential roles in peripheral nerve regeneration, but several drawbacks prevent their use for nerve repair. Adult stem cells from adipose tissue (ASC) or bone marrow (BM-MSC) can be differentiated into an SC-like phenotype and used as SC replacements. The aim of this study was to investigate GABA-B receptor functional expression in differentiated stem cells by assessing the similarity to SC. By means of RT-PCR and Western blot methodologies, BM-MSC and ASC were found to express both GABA-B1 and GABA-B2 receptor subunits. The expression levels of GABA-B1b and GABA-B2 receptors were influenced by SC-like differentiation, as shown by Western blot studies. GABA-B receptor stimulation with baclofen reduced the proliferation rate of SC and differentiated ASC (dASC) but not that of dBM-MSC. In conclusion, both of the subunits that assemble into a functional GABA-B receptor are present in differentiated stem cells. Furthermore, GABA-B receptors in dASC are functionally active, regulating a key process such as proliferation. The presence of functional GABA-B receptors on differentiated stem cells opens new opportunities for a possible pharmacological modulation of their physiology and phenotype.

Bone marrow- and adipose-derived stem cells show expression of myelin mRNAs and proteins.

AIMS: PNS myelin is formed by Schwann cells (SCs). In this study, we applied an in vitro model to study myelin formation, using bone marrow mesenchymal stem cells and adipose-derived stem cells differentiated into SC-like cells and co-cultured with dissociated adult dorsal root ganglia neurons. METHODS: Immunocytochemistry, reverse transcription-PCR and western blotting techniques were used to investigate the expression of myelin proteins at both the transcriptional and translational level. RESULTS: Transcripts for protein zero, peripheral myelin protein 22 and myelin basic protein were detected in differentiated stem cells following co-culture with neuronal cells. Furthermore, protein zero, peripheral myelin protein 22 and myelin basic proteins were recognized in the co-cultures. These results were consistent with immunostaining of myelin proteins and with observation by electron microscopy. CONCLUSION: Both types of adult stems cells differentiated into SC-like cells have potential to myelinate neuronal cells during regeneration, being functionally identical to SCs of the PNS.

N-acetylcysteine alters apoptotic gene expression in axotomised primary sensory afferent subpopulations.

Novel approaches are required in peripheral nerve injury management because current surgical techniques, which do not address axotomy-induced neuronal death, lead to deficient sensory recovery. Sensory neuronal death has functional preference with cutaneous neurons dying in great numbers whilst muscle afferents survive axotomy. This offers the potential of comparing similar cell types that suffer distinct fates upon nerve injury. Here, a novel approach, combining in vivo rat nerve injury model with laser microdissection and quantitative real-time polymerase chain reaction, identifies crucial disparities in apoptotic gene expression attributable to subpopulations of differing sensory modalities and examines the response to N-acetylcysteine (NAC) therapy. We show that axotomised muscle afferent neurons survive injury due to a neuroprotective response which markedly downregulates Bax and caspase-3 mRNA. In contrast, axotomised cutaneous sensory neurons significantly upregulate caspase-3 and alter both Bcl-2 and Bax expression such that pro-apoptotic Bax predominates. N-Acetylcysteine (NAC) intervention promotes neuroprotection of cutaneous sensory neurons through considerable upregulation of Bcl-2 and downregulation of both Bax and caspase-3 mRNA. The data presented identifies differential activation of apoptotic genes in axotomised neuronal subpopulations. Furthermore, NAC therapy instigates apoptotic gene expression changes in axotomised neurons, thereby offering pharmacotherapeutic potential in the clinical treatment of nerve injury.

Characterisation of human mesenchymal stem cells following differentiation into Schwann cell-like cells.

Cell-based therapies provide a clinically applicable and available alternative to nerve autografts. Our previous studies have characterised rat-derived mesenchymal stem cells (MSC) and here we have investigated the phenotypic, molecular and functional characteristics of human-derived MSC (hMSC) differentiated along a Schwann cell lineage. The hMSC were isolated from healthy human donors and the identity of the undifferentiated hMSC was confirmed by the detection of MSC specific cells surface markers. The hMSC were differentiated along a glial cell lineage using an established cocktail of growth factors including glial growth factor-2. Following differentiation, the hMSC expressed the key Schwann cell (SC) markers at both the transcriptional and translational level. More importantly, we show the functional effect of hMSC on neurite outgrowth using an in vitro co-culture model system with rat-derived primary sensory neurons. The number of DRG sprouting neurites was significantly enhanced in the presence of differentiated hMSC; neurite length and density (branching) were also increased. These results provide evidence that hMSC can undergo molecular, morphological and functional changes to adopt a SC-like behaviour and, therefore, could be suitable as SC substitutes for nerve repair in clinical applications.

Schwann cell mediated trophic effects by differentiated mesenchymal stem cells.

Mesenchymal stem cells were isolated from the bone marrow of rats and differentiated to provide a functional substitute for slow growing Schwann cells for peripheral nerve regeneration. To assess the properties of the differentiated mesenchymal stem cell, the cells were co-cultured with dorsal root ganglia and the secretion of the neurotrophic factors and the neurite outgrowth was evaluated. The neurite outgrowth of the dorsal root ganglia neurons was enhanced in co-culture with the differentiated stem cells compared to the undifferentiated stem cells. Differentiated stem cells like Schwann cells were responsible for the stimulation of longer and branched neurites. Using enzyme-linked immunosorbant assays and blocking antibodies, we have shown that this effect is due to the release of brain derived neurotrophic factor and nerve growth factor, which were up-regulated in differentiated mesenchymal stem cells following co-culture. The relevance of the tyrosine kinase receptors was confirmed by the selective tyrosine kinase inhibitor, K252a which abolished the neurite outgrowth of the dorsal root ganglia neurons when co-cultured with the differentiated mesenchymal stem cells similar to Schwann cells. The results of the study further support the notion that mesenchymal stem cells can be differentiated and display trophic influences as those of Schwann cells.

Growth factors in mesenchymal stem cells following glial-cell differentiation.

Schwann cells are essential facilitators of peripheral nerve regeneration following injury, as they provide physical support and guidance. In vitro these supporting cells are slow-growing and hence are not well suited to a tissue-engineering approach to nerve repair. We have differentiated rat bone-marrow-derived mesenchymal stem cells into Schwann-cell-like cells using a cocktail of growth factors, including glial growth factor-2. Qualitative reverse transcription-PCR, Western-blotting and immunocytochemical approaches were used to investigate the mRNA transcript levels and protein expression of glial cell markers and neurotrophic factors in differentiated mesenchymal stem cells compared with the levels found in Schwann cells (which acted as a positive control). The results showed that differentiated mesenchymal stem cells expressed transcripts and proteins for the specific glial growth receptor 2, erbB3 and neurotrophic factors, nerve growth factor, brain-derived neurotrophic factor, glial-derived neurotrophic factor and leukaemia inhibitory factor. Expression of these growth factors provides further evidence that differentiated mesenchymal stem cells appear to have cellular and molecular characteristics similar to those of Schwann cells.

Neurotrophins 3 and 4 differentially regulate NCAM, L1 and N-cadherin expression during peripheral nerve regeneration.

The addition of NT-3 (neurotrophin 3) or NT-4 to injured nerves improves their regeneration potential and may aid axon guidance. It is not well defined whether NTs (neurotrophins) influence other elements, such as the cell-adhesion molecules, which promote nerve guidance and regeneration. Using poly-3-hydroxybutyrate conduits, we applied either NT-3 or NT-4 to axotomized rat sciatic nerves and monitored nerve regeneration and cell-adhesion molecule expression. Regenerating nerves were stained with antibodies against NCAM (neural cell-adhesion molecule) and N-cadherin 2 weeks after injury and staining intensity was quantified. NCAM, N-cadherin and L1 (L1 cell-adhesion molecule) transcription was measured in the proximal and distal stumps and ipsilateral DRG (dorsal root ganglia) (fourth and fifth DRG) using RT (reverse transcriptase)-PCR. Both NT-3 and NT-4 increased NCAM and L1 transcript levels in the DRG of axotomized nerves. This is reflected in the increased NCAM expression at the proximal stump and regeneration front. Increased levels of NCAM were also observed in the distal stump. NT-4 administration increased N-cadherin levels proximal to the injury, but not distally. Following NT-3 administration, N-cadherin expression decreased in proximal and distal stumps compared with the control. In conclusion, NTs differentially alter adhesion molecule expression in regenerating nerves and transcription in the corresponding DRG, although these changes in expression do not alter NT-enhanced regeneration. Thus we propose that retrograde transport of the NTs to the DRG affects adhesion molecule transcription, reflected by protein expression in peripheral nerve axons.

Expression of PAX 3 alternatively spliced transcripts and identification of two new isoforms in human tumors of neural crest origin.

The developmental gene PAX 3 is expressed in the early embryo in developing muscle and elements of the nervous system, including the brain. Since no one has investigated the expression of the isoforms of PAX 3 in the neuroectodermal tumors melanoma and small cell lung cancer (SCLC), we have carried out a comprehensive screening for the expression of the isoforms PAX 3a-e using RT-PCR in human melanoma cell lines, primary human ocular and secondary cutaneous melanomas. We have identified 2 new isoforms of PAX 3, g and h, which we have isolated, cloned and sequenced. Sets of primers for each isoform were designed and their specificity was confirmed by sequence analysis of the products. The isoforms PAX 3a-e were detected in all human cutaneous melanoma cell lines (8/8), but only PAX 3c (1/2) and PAX 3d (2/2) in ocular melanoma cell lines. The same PAX 3 isoforms were detected in more than 80% of human cutaneous melanomas: PAX 3a and b (15/17), PAX 3c (14/17), PAX 3d (16/17) and PAX 3e (15/17). In contrast the results for 7 SCLC cell lines were PAX 3a (0/7), PAX 3b (1/7), PAX 3c (3/7), PAX 3d (6/7), PAX 3e (2/7); 8/8 cutaneous melanoma cell lines and 8/8 ocular melanoma tissues, together with 14/17 cutaneous melanoma tissues screened, expressed the new isoform PAX 3g. All 8 cutaneous melanoma cell lines expressed PAX 3h, but it was not detectable in any of the tumor tissues (0/20). Neither of the 2 ocular melanoma cell lines expressed the 2 new isoforms. Comparison of the different amplicon staining intensities on a gel suggests that PAX 3c and PAX 3d are the predominant transcripts expressed, with relatively low expression of PAX 3e and PAX 3h. We propose that these and the 2 new isoforms we have discovered may be important in oncogenesis and differential diagnosis of melanomas or SCLC.