Moxifloxacin releasing intraocular implant based on a cross-linked hyaluronic acid membrane.

Intraocular antibiotic delivery is an important technique to prevent bacterial infection after ophthalmic surgery, such as cataract surgery. Conventional drug delivery methods, such as antibiotic eye drops, have limitations for intraocular drug delivery due to the intrinsic barrier effect of the cornea. Therefore, frequent instillation of antibiotic eyedrops is necessary to reach a sufficient bactericidal concentration inside the eye. In this study, an intraocular implant, MXF-HA, that combines hyaluronic acid (HA) and moxifloxacin (MXF) was developed to increase the efficiency of intraocular drug delivery after surgery. MXF-HA is manufactured as a thin, transparent, yellow-tinted membrane. When inserted into the eye in a dry state, MXF-HA is naturally hydrated and settles in the eye, and the MXF contained therein is delivered by hydrolysis of the polymer over time. It was confirmed through in vivo experiments that MXF delivery was maintained in the anterior chamber of the eye at a concentration sufficient to inhibit Pseudomonas aeruginosa and Staphylococcus aureus for more than 5 days after implantation. These results suggest that MXF-HA can be utilized as a potential drug delivery method for the prevention and treatment of bacterial infections after ophthalmic surgery.

Development of a novel hyaluronic acid membrane for the treatment of ocular surface diseases.

Ocular surface diseases (OSD) can cause serious visual deterioration and discomfort. Commercial artificial tear solution containing hyaluronic acid (HA) show excellent biocompatibility and unique viscoelastic characteristics. Here, we developed a novel HA membrane (HAM) by chemical crosslinking using 1,4-butanediol diglycidyl ether for the effective treatment of OSDs. The main purpose of HAMs is to provide sustained release of HA to modulate the wound healing response in OSDs. The safety and efficacy of HAMs were investigated using primary cultured human corneal epithelial cells and various OSD rabbit models. In the dry state, the HAM is firm, transparent, and easy to manipulate. When hydrated, it swells rapidly with high water retention and over 90% transmission of visible light. Human corneal epithelial cells and rabbit eyes showed no toxic response to HAM. Addition of HAMs to the culture medium enhanced human corneal epithelial cell viability and expression of cell proliferation markers. Investigation of HAM wound healing efficacy using mechanical or chemical corneal trauma and conjunctival surgery in rabbits revealed that application of HAMs to the ocular surface enhanced healing of corneal epithelium and reduced corneal limbal vascularization, opacity and conjunctival fibrosis. The therapeutic potential of HAMs in various OSDs was successfully demonstrated.

Spatiotemporal Progression of Microcalcification in the Hippocampal CA1 Region following Transient Forebrain Ischemia in Rats: An Ultrastructural Study.

Calcification in areas of neuronal degeneration is a common finding in several neuropathological disorders including ischemic insults. Here, we performed a detailed examination of the onset and spatiotemporal profile of calcification in the CA1 region of the hippocampus, where neuronal death has been observed after transient forebrain ischemia. Histopathological examinations showed very little alizarin red staining in the CA1 pyramidal cell layer until day 28 after reperfusion, while prominent alizarin red staining was detected in CA1 dendritic subfields, particularly in the stratum radiatum, by 14 days after reperfusion. Electron microscopy using the osmium/potassium dichromate method and electron probe microanalysis revealed selective calcium deposits within the mitochondria of degenerating dendrites at as early as 7 days after reperfusion, with subsequent complete mineralization occurring throughout the dendrites, which then coalesced to form larger mineral conglomerates with the adjacent calcifying neurites by 14 days after reperfusion. Large calcifying deposits were frequently observed at 28 days after reperfusion, when they were closely associated with or completely engulfed by astrocytes. In contrast, no prominent calcification was observed in the somata of CA1 pyramidal neurons showing the characteristic features of necrotic cell death after ischemia, although what appeared to be calcified mitochondria were noted in some degenerated neurons that became dark and condensed. Thus, our data indicate that intrahippocampal calcification after ischemic insults initially occurs within the mitochondria of degenerating dendrites, which leads to the extensive calcification that is associated with ischemic injuries. These findings suggest that in degenerating neurons, the calcified mitochondria in the dendrites, rather than in the somata, may serve as the nidus for further calcium precipitation in the ischemic hippocampus.

Enhanced expression of the calcium-sensing receptor in reactive astrocytes following ischemic injury in vivo and in vitro.

We recently demonstrated that the G protein-coupled calcium-sensing receptor (CaSR) is associated with the pathogenesis of ischemic stroke and may be involved in vascular remodeling and astrogliosis. To further substantiate the involvement of CaSR in the astroglial reaction common to ischemic insults, we investigated the temporal and cell type-specific expression patterns of CaSR in the hippocampus after transient forebrain ischemia. CaSR was constitutively expressed in neurons of the pyramidal and granule cell layers, whereas increased CaSR immunoreactivity was observed in reactive astrocytes, but not in activated microglia or macrophages, in the CA1 region of the post-ischemic hippocampus. Astroglial induction of CaSR expression was evident on days 3-7 after reperfusion and appeared to increase progressively through day 28, at which time CaSR expression was prominent in astrocytes with a highly reactive hypertrophic phenotype and elevated levels of glial fibrillary acidic protein. This expression pattern was supported by results of immunoblot analyses. Furthermore, CaSR expression was upregulated in rat primary cortical astrocytes exposed to oxygen-glucose deprivation, which undergo reactive gliosis-like changes. Thus, our results demonstrate that selective and long-lasting astroglial induction of CaSR expression is a common characteristic of ischemic injury and suggest its involvement in the ischemia-induced astroglial reaction.

Increased expression of Slit2 and its receptors Robo1 and Robo4 in reactive astrocytes of the rat hippocampus after transient forebrain ischemia.

Slit2 is a secreted glycoprotein that was originally identified as a chemorepulsive factor in the developing brain; however, it was recently reported that Slit2 is associated with adult neuronal function including a variety of pathophysiological processes. To elucidate whether Slit2 is implicated in the pathophysiology of ischemic injury, we investigated the temporal changes and cellular localization of Slit2 and its predominant receptors, Robo1 and Robo4, for 28 days after transient forebrain ischemia. Slit2 and its receptors had similar overall expression patterns in the control and ischemic hippocampi. The ligand and receptors were constitutively expressed in hippocampal neurons in control animals; however, in animals with ischemic injury, their upregulation was detected in reactive astrocytes, but not in neurons or activated microglia, in the CA1 region. Astroglial induction of Slit2 and its receptors occurred by day 3 after reperfusion, and appeared to increase progressively until the final time point on day 28. Their temporal expression patterns overlapped with the time period in which reactive astrocytes undergo dynamic structural changes and appear hypertrophic in the ischemic hippocampus. The immunohistochemical data were consistent with the results of the immunoblot analyses, indicating that the expression of Slit2 and Robo increased progressively over the relatively long period of 28 days examined here. Collectively, these results suggest that Slit2/Robo signaling may be involved in regulating the astroglial reaction via autocrine or paracrine mechanisms in post-ischemic processes. Moreover, this may contribute to the dynamic morphological changes that occur in astrocytes in response to ischemic injury.

Expression of SOCS2 mRNA and protein in the ischemic core and penumbra after transient focal cerebral ischemia in rats.

The suppressor of cytokine signaling 2 (SOCS2) has been reported to be involved in astroglial reactions and adult neurogenesis in the ischemic hippocampus. To elucidate whether SOCS2 is implicated in the pathophysiology of stroke, we investigate spatiotemporal regulation and identification of cell phenotypes expressing SOCS2 after transient focal cerebral ischemia. Weak hybridization signals for SOCS2 mRNA were constitutively observed in striatal neurons and upregulation of SOCS2 mRNA was induced in association with nestin-positive cells in stroke-lesioned rats. Analysis of the characteristics and phenotypes of SOCS2/nestin double-labeled cells revealed spatial differences between infarct and peri-infarct areas. SOCS2/nestin double-labeled cells in the infarct area were associated with the vasculature and were highly proliferative. In contrast, the double-labeled cells in the peri-infarct area were indeed glial fibrillary acidic protein (GFAP)-positive reactive astrocytes forming the glial scar, although nestin-negative reactive astrocytes also exhibited weak SOCS2 expression. In addition, induction of SOCS2 expression was observed in Iba1-positive cells showing a macrophage-like phenotype with amoeboid morphology; these cells were predominantly localized in the infarct area. In the peri-infarct area, only a small proportion of Iba1-positive cells with the morphology of brain macrophages expressed SOCS2 and most activated stellate microglial cells with thick and short processes exhibited weak or negligible SOCS2 expression. Thus, our results revealed the phenotypic and functional heterogeneity of SOCS2-expressing cells within infarct and peri-infarct areas, suggesting the involvement of SOCS2 in astroglial reactions and activation/recruitment of brain macrophages and its potential role in perivascular progenitors/stem cells after ischemic stroke.

miR-34a inhibits differentiation of human adipose tissue-derived stem cells by regulating cell cycle and senescence induction.

MicroRNAs (miRNAs) are critical in the maintenance, differentiation, and lineage commitment of stem cells. Stem cells have the unique property to differentiate into tissue-specific cell types (lineage commitment) during cell division (self-renewal). In this study, we investigated whether miR-34a, a cell cycle-regulating microRNA, could control the stem cell properties of adipose tissue-derived stem cells (ADSCs). First, we found that the expression level of miR-34a was increased as the cell passage number was increased. This finding, however, was inversely correlated with our finding that the overexpression of miR-34a induced the decrease of cell proliferation. In addition, miR-34a overexpression decreased the expression of various cell cycle regulators such as CDKs (-2, -4, -6) and cyclins (-E, -D), but not p21 and p53. The cell cycle analysis showed accumulation of dividing cells at S phase by miR-34a, which was reversible by co-treatment with anti-miR-34a. The potential of adipogenesis and osteogenesis of ADSCs was also decreased by miR-34a overexpression, which was recovered by co-treatment with anti-miR-34a. The surface expression of stem cell markers including CD44 was also down-regulated by miR-34a overexpression as similar to that elicited by cell cycle inhibitors. miR-34a also caused a significant decrease in mRNA expression of stem cell transcription factors as well as STAT-3 expression and phosphorylation. Cytokine profiling revealed that miR-34a significantly modulated IL-6 and -8 production, which was strongly related to cellular senescence. These data suggest the importance of miR-34a for the fate of ADSCs toward senescence rather than differentiation.

Ultrastructural investigation of microcalcification and the role of oxygen-glucose deprivation in cultured rat hippocampal slices.

Intracellular calcium accumulation is associated with cell death in several neuropathological disorders including brain ischemia, but the exact mechanisms of calcification need to be clarified. We used organotypic hippocampal slice culture - cultures subjected to oxygen-glucose deprivation (OGD) mimicking the in vivo situation to investigate the events underlying ectopic calcification. Alizarin red staining indicating calcium deposition was observed in the cornu ammonis (CA)1 and dentate gyrus regions in control hippocampal slices despite no specific labeling for cell death markers. Electron microscopy using the osmium/potassium dichromate method revealed scattered degenerated cells throughout the normally appearing CA1 region. They contained electron-dense precipitates within mitochondria, and electron probe microanalysis confirmed that they were calcifying mitochondria. Selective calcium deposition was noted within, but not beyond, mitochondria in these mineralized cells. They showed ultrastructural features of non-necrotic, non-apoptotic cell death and retained their compact ultrastructure, even after the majority of mitochondria were calcified. Unexpectedly, no intracellular calcification was noted in necrotic CA1 pyramidal cells after OGD, and there was no progression of calcification in OGD-lesioned slices. In addition, mineralized cells in both control and OGD-lesioned slices were closely associated with or completely engulfed by astrocytes but not microglia. These astrocytes were laden with heterogeneous cytoplasmic inclusions that appeared to be related with their phagocytic activity. These data demonstrate that microcalcification specifically associated with mitochondria might lead to a novel type of cell death and suggest that astrocytes may be involved in the phagocytosis of these mineralized cells and possibly in the regulation of ectopic calcification.

Differential expression of the calcium-sensing receptor in the ischemic and border zones after transient focal cerebral ischemia in rats.

G-protein-coupled calcium-sensing receptor (CaSR) has been recently recognized as an important modulator of diverse cellular functions, beyond the regulation of systemic calcium homeostasis. To identify whether CaSR is involved in the pathophysiology of stroke, we studied the spatiotemporal regulation of CaSR protein expression in rats undergoing transient focal cerebral ischemia, which was induced by middle cerebral artery occlusion. We observed very weak or negligible immunoreactivity for CaSR in the striatum of sham-operated rats, as well as in the contralateral striatum of ischemic rats after reperfusion. However, CaSR expression was induced in the ischemic and border zones of the lesion in ischemic rats. Six hours post-reperfusion there was an upregulation of CaSR in the ischemic zone, which seemed to decrease after seven days. This upregulation preferentially affected some neurons and cells associated with blood vessels, particularly endothelial cells and pericytes. In contrast, CaSR expression in the peri-infarct region was prominent three days after reperfusion, and with the exception of some neurons, it was mostly located in reactive astrocytes, up to day 14 after ischemia. On the other hand, activated microglia/macrophages in both the ischemic and border zones were devoid of specific labeling for CaSR at any time point after reperfusion, despite their massive infiltration in both regions. Our results show heterogeneity in CaSR-positive cells within the ischemic and border zones, suggesting that CaSR expression is regulated in response to the altered extracellular ionic environment caused by ischemic injury. Thus, CaSR may have a multifunctional role in the pathophysiology of ischemic stroke, possibly in vascular remodeling and astrogliosis.

Expression of vascular endothelial growth factor-C (VEGF-C) and its receptor (VEGFR-3) in the glial reaction elicited by human mesenchymal stem cell engraftment in the normal rat brain.

To determine whether vascular endothelial growth factor-C (VEGF-C) and its receptor (VEGFR-3) are involved in the glial reaction elicited by transplanted mesenchymal stem cells (MSCs), we examined the cellular localization of VEGF-C and VEGFR-3 proteins in the striatum of adult normal rats that received bone marrow-derived human MSCs. The MSC grafts were infiltrated with activated microglia/macrophages and astrocytes over a 2-week period post-transplantation, which appeared to parallel the loss of transplanted MSCs. VEGF-C/VEGFR-3 was expressed in activated microglia/macrophages recruited to the graft site, where the induction of VEGF-C protein was rather late compared with that of its receptor. VEGF-C protein was absent or very weak on day 3, whereas VEGFR-3 immunoreactivity was evident within the first three days. Furthermore, within three days, VEGF-C could be detected in the brain macrophages localized immediately adjacent to the needle track. At the same time, almost all the brain macrophages in both regions expressed VEGFR-3. Reactive astrocytes at the graft site expressed VEGFR-3, but not VEGF-C. These data demonstrated the characteristic time- and cell-dependent expression patterns for VEGF-C and VEGFR-3 within the engrafted brain tissue, suggesting that they may contribute to neuroinflammation in MSC transplantation, possibly through the recruitment and/or activation of microglia/macrophages and astrogliosis.