Downregulated Calcium-Binding Protein S100A16 and HSP27 in Placenta-Derived Multipotent Cells Induce Functional Astrocyte Differentiation.

Little is known about genes that induce stem cells differentiation into astrocytes. We previously described that heat shock protein 27 (HSP27) downregulation is directly related to neural differentiation under chemical induction in placenta-derived multipotent stem cells (PDMCs). Using this neural differentiation cell model, we cross-compared transcriptomic and proteomic data and selected 26 candidate genes with the same expression trends in both omics analyses. Those genes were further compared with a transcriptomic database derived from Alzheimer's disease (AD). Eighteen out of 26 candidates showed opposite expression trends between our data and the AD database. The mRNA and protein expression levels of those candidates showed downregulation of HSP27, S100 calcium-binding protein A16 (S100A16) and two other genes in our neural differentiation cell model. Silencing these four genes with various combinations showed that co-silencing HSP27 and S100A16 has stronger effects than other combinations for astrocyte differentiation. The induced astrocyte showed typical astrocytic star-shape and developed with ramified, stringy and filamentous processes as well as differentiated endfoot structures. Also, some of them connected with each other and formed continuous network. Immunofluorescence quantification of various neural markers indicated that HSP27 and S100A16 downregulation mainly drive PDMCs differentiation into astrocytes. Immunofluorescence and confocal microscopic images showed the classical star-like shape morphology and co-expression of crucial astrocyte markers in induced astrocytes, while electrophysiology and Ca2+ influx examination further confirmed their functional characteristics. In conclusion, co-silencing of S100A16 and HSP27 without chemical induction leads to PDMCs differentiation into functional astrocytes.

Promotion of the Differentiation of Dental Pulp Stem Cells into Oligodendrocytes by Knockdown of Heat Shock Protein 27.

Stem cell-based therapy has been evaluated in many different clinical trials for various diseases. This capability was applied in various neurodegenerative diseases, such as multiple sclerosis, which is characterized by demyelination, axonal injury, and neuronal loss. Dental pulp stem cells (DPSCs) are mesenchymal stem cells from the oral cavity that have been studied with potential application for the regeneration of different tissues. Heat shock protein 27 (HSP27) regulates neurogenesis in the process of neural differentiation of placenta multipotent stem cells. Here, we hypothesize that HSP27 expression is also critical for the neural differentiation of DPSCs. An evaluation of the possible role of HSP27 in the differentiation of DPSCs was performed using gene knockdown and neural immunofluorescent staining. We found that HSP27 played a role in the differentiation of DPSCs and that knockdown of HSP27 in DPSCs rendered cells to oligodendrocyte progenitors; i.e., small hairpin specific for HSP27 DPSCs exhibited NG2-positive immunoreactivity and gave rise to oligodendrocytes or type-2 astrocytes. This neural differentiation of DPSCs may have clinical significance in the treatment of patients with neurodegenerative diseases. In conclusion, our data provide an example of the oligodendrocyte differentiation of a DPSC model, which may be applied in human regenerative medicine.

Classification of Activated Microglia by Convolutional Neural Networks.

Microglia are the resident macrophages in the central nervous system. Brain injuries, such as traumatic brain injury, hypoxia, and stroke, can induce inflammatory responses accompanying microglial activation. The morphology of microglia is notably diverse and is one of the prominent manifestations during activation. In this study, we proposed to detect the activated microglia in immunohistochemistry images by convolutional neural networks (CNN). 2D Iba1 images (40µm) were acquired from a control and a cardiac arrest treated Sprague-Dawley rat brain by a scanning microscope using a 20X objective. The training data were a collection of 54,333 single-cell images obtained from the cortex and midbrain areas, and curated by experienced neuroscientists. Results were compared between CNNs with different architectures, including Resnet18, Resnet50, Resnet101, and support vector machine (SVM) classifiers. The highest model performance was found by Resnet18, trained after 120 epochs with a classification accuracy of 95.5%. The findings indicate a potential application for using CNN in quantitative analysis of microglial morphology over regional difference in a large brain section.

Interleukin-1 receptor antagonist ameliorates the pain hypersensitivity, spinal inflammation and oxidative stress induced by systemic lipopolysaccharide in neonatal rats.

Perinatal inflammation-induced reduction in pain threshold may alter pain sensitivity to hyperalgesia or allodynia which may persist into adulthood. In this study, we investigated the anti-inflammatory protective effect of interleukin-1 receptor antagonist (IL-1ra), an anti-inflammatory cytokine, on systemic lipopolysaccharide (LPS)-induced spinal cord inflammation and oxidative stress, thermal hyperalgesia, and mechanical allodynia in neonatal rats. Intraperitoneal (i.p.) injection of LPS (2 mg/kg) or sterile saline was performed in postnatal day 5 (P5) rat pups, and IL-1ra (100 mg/kg) or saline was administered (i.p.) 5 min after LPS injection. Pain reflex behavior, spinal cord inflammation and oxidative stress were examined 24 h after LPS administration. Systemic LPS exposure led to a reduction of tactile threshold in the von Frey filament tests (mechanical allodynia) and pain response latency in the tail-flick test (thermal hyperalgesia) of P6 neonatal rats. Spinal cord inflammation was indicated by the increased numbers of activated glial cells including microglia (Iba1+) and astrocytes (GFAP+), and elevated levels of pro-inflammatory cytokine interleukin-1ß (IL-1ß), cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2) 24 h after LPS treatment. LPS treatment induced spinal oxidative stress as evidenced by the increase in thiobarbituric acid reactive substances (TBARS) content in the spinal cord. LPS exposure also led to a significant increase in oligodendrocyte lineage population (Olig2+) and mature oligodendrocyte cells (APC+) in the neonatal rat spinal cord. IL-1ra treatment significantly reduced LPS-induced effects including hyperalgesia, allodynia, the increased number of activated microglia, astrocytes and oligodendrocytes, and elevated levels of IL-1ß, COX-2, PGE2, and lipid peroxidation (TBARS) in the neonatal rat spinal cord. These data suggest that IL-1ra provides a protective effect against the development of pain hypersensitivity, spinal cord inflammation and oxidative stress in the neonatal rats following LPS exposure, which may be associated with the blockade of LPS-induced pro-inflammatory cytokine IL-1ß.

Hyperhomocysteinemia Causes Chorioretinal Angiogenesis with Placental Growth Factor Upregulation.

Hyperhomocysteinemia is a risk factor for atherosclerosis, which may also be associated with retinal vascular disease, diabetic retinopathy, retinal vein occlusion, and glaucoma. For this study, we established a hyperhomocysteinemia animal model to explore homocysteine (hcy)-related choroidal angiogenesis and possible related factors. We injected Sprague Dawley (SD) rats with different concentrations of hcy and performed color fundus imaging, fluorescein angiography, image-guided optical coherence tomography, and retinal histology to observe the retinal and choroidal changes. Subsequently, we observed prominent choroidal vasculature with congested and tortuous retinal and choroidal vessels in fundus angiographies of the hyperhomocysteinemia animal model. In the histological study, the choroidal capillaries proliferated in the hcy-treated eyes, mimicking choroidal neovascularization. Disrupted retinal pigment epithelium (RPE), abnormal branching vascular network (BVN), and polyp-like structures were also observed in the hcy-treated eyes. Furthermore, we found that placental growth factor (PlGF), but not vascular epithelial growth factor (VEGF), was the key mediating factor of this phenomenon. Our findings suggest that hyperhomocysteinemia might cause choroidal angiogenesis.

Systemic Lipopolysaccharide-Induced Pain Sensitivity and Spinal Inflammation Were Reduced by Minocycline in Neonatal Rats.

In this study, we investigated the effects of minocycline, a putative suppressor of microglial activation, on systemic lipopolysaccharide (LPS)-induced spinal cord inflammation, allodynia, and hyperalgesia in neonatal rats. Intraperitoneal (i.p.) injection of LPS (2 mg/kg) or sterile saline was performed in postnatal day 5 (P5) rat pups and minocycline (45 mg/kg) or vehicle (phosphate buffer saline; PBS) was administered (i.p.) 5 min after LPS injection. The von Frey filament and tail-flick tests were performed to determine mechanical allodynia (a painful sensation caused by innocuous stimuli, e.g., light touch) and thermal hyperalgesia (a condition of altered perception of temperature), respectively, and spinal cord inflammation was examined 24 h after the administration of drugs. Systemic LPS administration resulted in a reduction of tactile threshold in the von Frey filament tests and pain response latency in the tail-flick test of neonatal rats. The levels of microglia and astrocyte activation, pro-inflammatory cytokine interleukin-1ß (IL-1ß), cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2) in the spinal cord of neonatal rats were increased 24 h after the administration of LPS. Treatment with minocycline significantly attenuated LPS-induced allodynia, hyperalgesia, the increase in spinal cord microglia, and astrocyte activation, and elevated levels of IL-1ß, COX-2, and PGE2 in neonatal rats. These results suggest that minocycline provides protection against neonatal systemic LPS exposure-induced enhanced pain sensitivity (allodynia and hyperalgesia), and that the protective effects may be associated with its ability to attenuate LPS-induced microglia activation, and the levels of IL-1ß, COX-2, and PGE2 in the spinal cord of neonatal rats.

The Role of Gene Editing in Neurodegenerative Diseases.

Neurodegenerative diseases (NDs), at least including Alzheimer's, Huntington's, and Parkinson's diseases, have become the most dreaded maladies because there are no precise diagnostic tools or definite treatments for these debilitating diseases. The increased prevalence and a substantial impact on the social-economic and medical care of NDs propel governments to develop policies to counteract the impact. Although the etiologies of NDs are still unknown, growing evidence suggests that genetic, cellular, and circuit alternations may cause the generation of abnormal misfolded proteins, which uncontrolledly accumulate to damage and eventually overwhelm the protein-disposal mechanisms of these neurons, leading to a common pathological feature of NDs. If the functions and the connectivity can be restored, alterations and accumulated damages may improve. The gene-editing tools including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats-associated nucleases (CRISPR/CAS) have emerged as a novel tool not only for generating specific ND animal models for interrogating the mechanisms and screening potential drugs against NDs but also for the editing sequence-specific genes to help patients with NDs to regain function and connectivity. This review introduces the clinical manifestations of three distinct NDs and the applications of the gene-editing technology on these debilitating diseases.

The Neuroprotective and antioxidative effects of submicron and blended Lycium barbarum in experimental retinal degeneration in rats.

The object is to determine the neuroprotective and antioxidative effects of submicron and blended Lycium barbarum (LB) on retinal degeneration as evaluated by ERG, retinal histopathology and assays of antioxidant (total GSH) and peroxidant (MDA) in the retina. A rat model of light-induced retinal degeneration was used to assess the protective effect of different forms of Lycium barbarum (LB) on retinal degeneration. Rats were divided into four experimental groups, normal control, light-induced untreated, submicron LB and blended LB treated. The rats of submicron and blended groups were treated with 250 mg/kg LB orally once daily for 54 days, followed by induction of retinal degeneration. Retinal function was assessed by electroretinography (ERG). Enzyme-linked immunosorbent assay of the retina lysates was measured for the levels of antioxidants, reduced glutathione and glutathione disulfide, and peroxidants, malondialdehyde, in the retina. The ERG results showed a protective effect in LB treated groups with a greater effect observed in submicron LB treated group than the blended LB treated group. There were higher levels of GSH plus GSSG and lower MDA in submicron LB treated group than other groups. In conclusion, LB provided protective and antioxidative effects on the rat retina with light-induced retinal degeneration. Submicron LB protected degenerative retina better than blended LB. LB is effective against oxidative stress in the degenerative retina.

Neuroprotective Effects of Intranasal IGF-1 against Neonatal Lipopolysaccharide-Induced Neurobehavioral Deficits and Neuronal Inflammation in the Substantia Nigra and Locus Coeruleus of Juvenile Rats.

Neonatal lipopolysaccharide (LPS) exposure-induced brain inflammation resulted in motor dysfunction and brain dopaminergic neuronal injury, and increased the risks of neurodegenerative disorders in adult rats. Our previous studies showed that intranasal administration of insulin-like growth factor-1 (IGF-1) protects against LPS-induced white matter injury in the developing rat brain. To further examine whether IGF-1 protects against LPS-induced brain neuronal injury and neurobehavioral dysfunction, recombinant human IGF-1 (rhIGF-1) at a dose of 50 µg/pup was administered intranasally 1 h following intracerebral injection of LPS (1 mg/kg) in postnatal day 5 (P5) Sprague-Dawley rat pups. Neurobehavioral tests were carried out from P7 to P21, and brain neuronal injury was examined at P21. Our results showed that LPS exposure resulted in disturbances of motor behaviors in juvenile rats. Moreover, LPS exposure caused injury to central catecholaminergic neurons, as indicated by a reduction of tyrosine hydroxylase (TH) immunoreactivity in the substantia nigra (SN), ventral tegmental area (VTA) and olfactory bulb (OB), and brain noradrenergic neurons, as indicated by a reduction of TH immunoreactivity in the locus coeruleus (LC) of the P21 rat brain. The LPS-induced reduction of TH+ cells was observed at a greater degree in the SN and LC of the P21 rat brain. Intranasal rhIGF-1 treatment attenuated LPS-induced central catecholaminergic neuronal injury and motor behavioral disturbances, including locomotion, beam walking test and gait analysis. Intranasal rhIGF-1 administration also attenuated LPS-induced elevation of IL-1ß levels and numbers of activated microglia, and cyclooxygenase-2+ cells, which were double labeled with TH+ cells in the SN, VTA, OB and LC of the P21 rat brain. These results suggest that IGF-1 may provide protection against neonatal LPS exposure-induced central catecholaminergic neuronal injury and motor behavioral disturbances, and that the protective effects are associated with the inhibition of microglia activation and the reduction of neuronal oxidative stress by the suppression of the neuronal cyclooxygenase-2 expression.

Homocysteine Facilitates Prominent Polygonal Angiogenetic Networks of a Choroidal Capillary Sprouting Model.

Purpose: To investigate the effects of homocysteine on choroidal angiogenesis, we established an ex vivo choroidal sprouting explant model and examined the potential growth factors for angiogenesis. Methods: Choroid fragments with retinal pigment epithelium were isolated from mouse and embedded in Matrigel. Homocysteine at different concentrations were added to the culture mediums. The choroidal explants were observed at different time points, and the total area of choroidal sprouting was measured and analyzed. Results: Homocysteine evoked choroidal capillary sprouting by inducing capillary endothelial cell proliferation with pericyte formation and by facilitating polygonal angiogenetic networks. In some cases, vascular lumens were observed in the newly forming capillaries facilitated by homocysteine. The choroidal sprouting effect of homocysteine can only be observed at a certain range of homocysteine concentration, with 1-mM homocysteine exhibiting the most significantly increased choroidal sprouting areas. Isolectin overexpression was noted in the homocysteine-treated group. Possible growth factors for angiogenesis were detected through immunofluorescent staining, which demonstrated the overexpression of platelet-derived growth factor C and angiopoietin 1 in the homocysteine-treated preparations only. In these preparations, platelet-derived growth factor C was highly expressed in the tip cells of sprouting capillaries. Conclusions: We therefore conclude that platelet-derived growth factor C and angiopoietin 1 may play key roles in the choroid angiogenesis evoked by homocysteine.

Suppression of HSP27 Restores Retinal Function and Protects Photoreceptors From Apoptosis in a Light-Induced Retinal Degeneration Animal Model.

Purpose: We used a light-induced retinal degeneration animal model to investigate possible roles of heat shock protein 27 (HSP27) in retinal/photoreceptor protection. Methods: Sprague-Dawley rats were used for the light-induced retinal degeneration animal model. The histology of eye sections was observed for morphologic changes in the retina. Cell apoptosis was examined in each group using the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and electroretinography was used to evaluate retinal function. Protein and mRNA expression levels of different retinal cell markers were also detected through immunofluorescence staining, Western blotting, and real-time PCR. Results: The thickness of the outer nuclear layer significantly decreased after 7-day light exposure. Moreover, we injected a viral vector for silencing HSP27 expression into the eyes and observed that photoreceptors were better preserved in the HSP27-suppressed (sHSP27) retina 2 weeks after injection. HSP27 suppression also reduced retinal cell apoptosis caused by light exposure. In addition, the loss of retinal function caused by light exposure was reversed on suppressing HSP27 expression. We subsequently found that the expression of the Rho gene and immunofluorescence staining of rhodopsin and arrestin (cell markers for photoreceptors) increased in sHSP27-treated retinas. HSP27 suppression did not affect the survival of ganglion and amacrine cells. Conclusions: Retinal cell apoptosis and functional loss were observed after 7-day light exposure. However, in the following 2 weeks after light exposure, HSP27 suppression may initiate a protective effect for retinal cells, particularly photoreceptors, from light-induced retinal degeneration.

Knocking down of heat-shock protein 27 directs differentiation of functional glutamatergic neurons from placenta-derived multipotent cells.

This study presents human placenta-derived multipotent cells (PDMCs) as a source from which functional glutamatergic neurons can be derived. We found that the small heat-shock protein 27 (HSP27) was downregulated during the neuronal differentiation process. The in vivo temporal and spatial profiles of HSP27 expression were determined and showed inverted distributions with neuronal proteins during mouse embryonic development. Overexpression of HSP27 in stem cells led to the arrest of neuronal differentiation; however, the knockdown of HSP27 yielded a substantially enhanced ability of PDMCs to differentiate into neurons. These neurons formed synaptic networks and showed positive staining for multiple neuronal markers. Additionally, cellular phenomena including the absence of apoptosis and rare proliferation in HSP27-silenced PDMCs, combined with molecular events such as cleaved caspase-3 and the loss of stemness with cleaved Nanog, indicated that HSP27 is located upstream of neuronal differentiation and constrains that process. Furthermore, the induced neurons showed increasing intracellular calcium concentrations upon glutamate treatment. These differentiated cells co-expressed the N-methyl-D-aspartate receptor, vesicular glutamate transporter, and synaptosomal-associated protein 25 but did not show expression of tyrosine hydroxylase, choline acetyltransferase or glutamate decarboxylase 67. Therefore, we concluded that HSP27-silenced PDMCs differentiated into neurons possessing the characteristics of functional glutamatergic neurons.

Effect of a complex lutein formula in an animal model for light-induced retinal degeneration.

Several retinal degenerative diseases cause vision loss and retinal cell death. Currently, people face prolonged exposure to digital screens, rendering vision protection from light exposure a critical topic. In this study, we designed a complex lutein formula (CLF) by combining several natural compounds: Calendula officinalis, Lycium barbarum, Vaccinium myrtillus, Cassia obtusifolia, and Rhodiola rosea. In addition, we evaluated the protective effects of the formula on retinal functions in an animal model for light-induced retinal degeneration. We employed electroretinography to analyse retinal function, and conducted a histological examination of the morphological changes in the retina treated under various conditions. We revealed that the retinal function in animals exposed to light for 7 days decreased significantly; however, the retinal function of animals that had received the CLF exhibited superior performance, despite light exposure. In addition, a greater portion of the outer nuclear layer (ONL) (i.e. the nuclei of photoreceptors) in these animals was preserved compared with the animals that had not received the formula after 7 days of light exposure. These results revealed that our dietary CLF supplement attenuated retinal function loss resulting from long-term light exposure.

Protein kinase C-dependent growth-associated protein 43 phosphorylation regulates gephyrin aggregation at developing GABAergic synapses.

Growth-associated protein 43 (GAP43) is known to regulate axon growth, but whether it also plays a role in synaptogenesis remains unclear. Here, we found that GAP43 regulates the aggregation of gephyrin, a pivotal protein for clustering postsynaptic GABA(A) receptors (GABA(A)Rs), in developing cortical neurons. Pharmacological blockade of either protein kinase C (PKC) or neuronal activity increased both GAP43-gephyrin association and gephyrin misfolding-induced aggregation, suggesting the importance of PKC-dependent regulation of GABAergic synapses. Furthermore, we found that PKC phosphorylation-resistant GAP43(S41A), but not PKC phosphorylation-mimicking GAP43(S41D), interacted with cytosolic gephyrin to trigger gephyrin misfolding and its sequestration into aggresomes. In contrast, GAP43(S41D), but not GAP43(S41A), inhibited the physiological aggregation/clustering of gephyrin, reduced surface GABA(A)Rs under physiological conditions, and attenuated gephyrin misfolding under transient oxygen-glucose deprivation (tOGD) that mimics pathological neonatal hypoxia. Calcineurin-mediated GAP43 dephosphorylation that accompanied tOGD also led to GAP43-gephyrin association and gephyrin misfolding. Thus, PKC-dependent phosphorylation of GAP43 plays a critical role in regulating postsynaptic gephyrin aggregation in developing GABAergic synapses.

Reevaluating reference ranges of oxygen saturation for healthy full-term neonates using pulse oximetry.

BACKGROUND: We compared our clinical experience with currently available reference oxygen saturation level (SpO(2)) values from the American Academy of Pediatrics/American Heart Association (AAP/AHA) neonatal resuscitation program guidelines. METHODS: We enrolled 145 healthy full-term neonates; infants showing respiratory distress and those with serious congenital anomalies were excluded. SpO(2) values at every 1 minute until 10 minutes after birth were measured and recorded. Infants were classified into the cesarean section (CS) and normal spontaneous delivery (NSD) groups for evaluating differences. The 10(th) percentiles of SpO(2) at each minute were used as the lower limits of normal oxygen saturation, and these were compared with the lowest target values recommended in the AAP/AHA guidelines. RESULTS: Overall, 130 vigorous full-term neonates (median gestational age: 38 5/7 weeks; body weight at birth: 2405-3960 g) were analyzed. The median SpO(2) were 67% and 89% at the 1(st) and 4(th) minute, respectively. On average, SpO(2) values reached >90% at the 5(th) minute. No statistical differences were noted in the SpO(2) values between the CS and NSD groups after 5 minutes; however, a trend of higher SpO(2) was observed in the NSD group. We noted a gradually increasing trend for SpO(2) values over time, similar to that noted in the AAP/AHA guidelines. However, SpO(2) values at the 10(th) percentiles of each minute within the first 5 minutes in our study were equal to or significantly lower than those in the AAP/AHA guidelines; moreover, at the 10(th) minute, SpO(2) values at the 10(th) percentiles were significantly higher than those in the guidelines. CONCLUSION: The delivery modes did not affect the SpO(2) values of full-term healthy neonates. Discrepancies in SpO(2) changes in full-term neonates not requiring resuscitation between this study and the AAP/AHA guidelines were significant. SpO(2) ranges for each time point within the first 10 minutes after birth should therefore be reevaluated locally.

Effect of prenatal methadone on reinstated behavioral sensitization induced by methamphetamine in adolescent rats.

It has been known that methadone maintenance treatment is the standard treatment of choice for pregnant opiate addicts. However, there are few data on newborn outcomes especially in the cross talk with other addictive agents. The present study was to investigate the effect of prenatal exposure to methadone on methamphetamine (METH)-induced behavioral sensitization as an indicator of drug addiction in later life. Pregnant rats received saline or methadone (7 mg/kg, s.c.) twice daily from E3 to E20. To induce behavioral sensitization, offspring (5 weeks old) were treated with METH (1mg/kg, i.p.) or saline once daily for 5 consecutive days. Ninety-six hours (day 9) after the 5th treatment with METH or saline, animals received a single dose of METH (1mg/kg, i.p.) or saline to induce the reinstated behavioral sensitization. Prenatal methadone treatment enhanced the level of development of locomotor behavioral sensitization to METH administration in adolescent rats. Prenatal methadone treatment also enhanced the reinstated locomotor behavioral sensitization in adolescent rats after the administration had ceased for 96 h. These results indicate that prenatal methadone exposure produces a persistent lesion in the dopaminergic system, as indicated by enhanced METH-induced locomotor behavioral sensitization (before drug abstinence) and reinstated locomotor behavioral sensitization (after short term drug abstinence) in adolescent rats. These findings show that prenatal methadone exposure may enhance susceptibility to the development of drug addiction in later life. This could provide a reference for drug usage such as methamphetamine in their offspring of pregnant woman who are treating with methadone.

Evaluation of potential topical and systemic neuroprotective agents for ocular hypertension-induced retinal ischemia-reperfusion injury.

OBJECTIVE: To evaluate for drugs with superior neuroprotective efficacy and investigate their underlying mechanisms related to antioxidation. PROCEDURES: Brinzolamide (1%), timolol (0.5%), minocycline (22 mg/kg), lidocaine (1.5 mg/kg), and methylprednisolone (30 mg/kg) were administered to Sprague-Dawley (SD) rats. The retina was evaluated by electroretinography and histological analysis. The antioxidative capacity of drugs was evaluated to clarify the underlying mechanism. The oxidant/antioxidant profiles of plasma, red blood cells, and retina were analyzed by lipid peroxidation (malondialdehyde) and by measuring the activities of antioxidants. Proteomic analysis was used to investigate the possible protective mechanisms of the drug against ischemia-reperfusion injury. RESULTS: The results suggested that timolol, methylprednisolone, and minocycline protected retinal function. Methylprednisolone and minocycline possessed good antioxidative activity. Brinzolamide and lidocaine preserved the structural integrity of the retina, but not retinal function. CONCLUSION: Methylprednisolone, minocycline, and timolol have potential acute or delayed benefit in retinal ischemia-reperfusion injury. Their neuroprotective actions depend at least partially on the ability to alleviate oxidative stress.

Involvement of SDF1a and STAT3 in granulocyte colony-stimulating factor rescues optic ischemia-induced retinal function loss by mobilizing hematopoietic stem cells.

PURPOSE: Granulocyte colony-stimulating factor (G-CSF) has been applied clinically for several years. In this study, we used G-CSF to induce the mobilization of hematopoietic progenitor cells into peripheral blood in an ischemia-induced retinal degeneration model. METHODS: Male Sprague-Dawley rats received G-CSF treatment for 5 days following optic ligation. Histologic and functional evaluations were performed and results were compared with those from untreated rats. Real-time PCR, Western blotting, and immunohistochemical analyses were used to evaluate the expression of retinal cell markers and other substances. RESULTS: Retinal histology showed that transient optic ligation induced retinal cell loss. Postischemia, animals that received G-CSF treatment had a higher retinal cell survival rate than that of control animals. Analysis of apoptosis showed that retinas from G-CSF-treated animals exhibited fewer apoptotic cells than those from control retinas. Immunoblotting analyses indicated the presence of greater numbers of CD34-, but less chemokine receptor type 4 (CXCR4)-, and stromal cell-derived factor 1 alpha (SDF1α)-positive cells in the G-CSF-treated ischemic retinas than in ischemic retinas without treatment 14 days after ischemia. The ischemic retinas from G-CSF-treated animals displayed upregulated Thy1 and opsin expression compared with the retinas from untreated animals. Electroretinography indicated superior retinal function in animals treated with G-CSF than in untreated animals postischemia, and that STAT3 might play an important role. CONCLUSIONS: Our results suggest that G-CSF reduces optic ischemia-induced retinal cell loss, possibly through STAT3-regulated mobilization of hematopoietic progenitor cells to the retina.

Mu-opioid receptor knockout mice are more sensitive to chlordiazepoxide-induced anxiolytic behavior.

We have previously demonstrated benzodiazepine binding in the cortex and hippocampus of mu-opioid receptor knockout (KO) mice. It is known that benzodiazepine receptors are involved in regulating anxiety-like behaviors. Thus, the present study was designed to examine whether there are changes in anxiety-like behavior in mice lacking mu-opioid receptors. To produce anxiolytic activity (less anxiety), the prototype benzodiazepine receptor agonist chlordiazepoxide (CDP, 5 mg/kg) was intraperitoneally administered in wild type (WT) and mu-opioid receptor KO mice. We found that compared to WT mice, mu-opioid receptor KO mice showed enhanced anxiolytic activity to CDP, including increased number of entries into open arm, increased percentage of the time spent in open arms, and decreased percentage of the time spent in enclosed arms in the elevated plus-maze test. We also assessed protein expression of the gamma-aminobutyric acid (GABA) synthetic enzyme (glutamic acid decarboxylase; GAD). Western blotting data indicated that neither the lack of mu-opioid receptors nor CDP treatment altered cortical or hippocampal GAD65 or GAD67 protein expression. These data indicate that compared with WT, mu-opioid receptor KO mice experienced less anxiety and exhibited enhanced anxiolytic activity to CDP treatment, and these effects were not dependent on GAD65 or GAD67 protein expression. Our previous and present data suggest that the anxiolytic activity displayed in mu-opioid receptor KO mice is associated with upregulation of the benzodiazepine receptor system.