Adipose mesenchymal stem cell-derived extracellular vesicles reduce glutamate-induced excitotoxicity in the retina.

Adipose mesenchymal stem cells (ADSCs) have protective effects against glutamate-induced excitotoxicity, but ADSCs are limited in use for treatment of optic nerve injury. Studies have shown that the extracellular vesicles (EVs) secreted by ADSCs (ADSC-EVs) not only have the function of ADSCs, but also have unique advantages including non-immunogenicity, low probability of abnormal growth, and easy access to target cells. In the present study, we showed that intravitreal injection of ADSC-EVs substantially reduced glutamate-induced damage to retinal morphology and electroretinography. In addition, R28 cell pretreatment with ADSC-EVs before injury inhibited glutamate-induced overload of intracellular calcium, downregulation of α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor (AMPAR) subunit GluA2, and phosphorylation of GluA2 and protein kinase C alpha in vitro. A protein kinase C alpha agonist, 12-O-tetradecanoylphorbol 13-acetate, inhibited the neuroprotective effects of ADSC-EVs on glutamate-induced R28 cells. These findings suggest that ADSC-EVs ameliorate glutamate-induced excitotoxicity in the retina through inhibiting protein kinase C alpha activation.

The efficacy and safety of using pyrotinib combined with capecitabine as neoadjuvant therapy in elderly patients with HER2-positive breast cancer: a single-arm prospective clinical trial.

Background: Pyrotinib combined with capecitabine has been approved for the treatment of patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer in China. To date, the management of early-stage or locally advanced HER2-positive breast cancer in the clinic remains challenging. We conducted this trial to investigate the efficacy and safety of pyrotinib combined with capecitabine as neoadjuvant therapy (NAT) in elderly patients with HER2-positive breast cancer. Due to the stimulation of blood vessels by chemotherapy drugs, the elasticity of blood vessels in the elderly decreases, and then chemotherapy infusion is more likely to lead to phlebitis. Both pyrotinib and capecitabine can be taken to facilitate home treatment for elderly patients with HER2-positive breast cancer (BC). Methods: From January 2020 to March 2021, patients aged between 70 and 81 years old with stage IIA-IIIB HER2-positive breast cancer were screened, enrolled, and assigned to receive six cycles of pyrotinib (320-400 mg, orally, once daily) plus capecitabine (1,250 mg/m2, orally, twice daily) on days 1-14 in every 21-day cycle. The primary endpoint was the objective response rate (ORR). Adverse events (AEs) were assessed in every neoadjuvant cycle. Surgery was performed after the last cycle, and the total pathological complete response (tpCR) was evaluated postoperatively. Results: Of the 23 patients enrolled, the ORR was 100% (23/23; 95% confidence intervals: 85 to 100). All patients underwent surgery with a tpCR rate of 43.5% (10/23; 95% confidence intervals: 23 to 66). The most common AE was diarrhea, occurring in 19 of 23 patients (82.6%); most of these patients sustained mild diarrhea (Grade 1 or Grade 2) and only three had moderate diarrhea (Grade 3). The incidences of other AEs, including weakness, loss of appetite, leukopenia, nausea, vomiting, hand-foot syndrome, etc., were low and the symptoms were mild. No severe AEs (Grade 4 or 5) were observed throughout the treatment. Conclusion: In our study, pyrotinib combined with capecitabine as neoadjuvant therapy in elderly women with HER2-positive breast cancer is safe and showed efficacy in this population, which may be widely used as a protocol for clinical neoadjuvant therapy.

Chronic corticosterone administration reduces dendritic complexity in mature, but not young granule cells in the rat dentate gyrus.

BACKGROUND: Our previous work has shown that exposure to the stress hormone corticosterone (40 mg/kg CORT) for two weeks induces dendritic atrophy of pyramidal neurons in the hippocampal CA3 region and behavioral deficits. However, it is unclear whether this treatment also affects the dentate gyrus (DG), a subregion of the hippocampus comprising a heterogeneous population of young and mature neurons. OBJECTIVE: We examined the effect of CORT treatment on the dendritic complexity of mature and young granule cells in the DG. METHODS: We utilized a Golgi staining method to investigate the dendritic morphology and spine density of young neurons in the inner granular cell layer (GCL) and mature neurons in the outer GCL in response to CORT application. The expressions of glucocorticoid receptors during neuronal maturation were examined using Western blot analysis in a primary hippocampal neuronal culture. RESULTS: Sholl analysis revealed that CORT treatment decreased the number of intersections and shortened the dendritic length in mature, but not young, granule cells. However, the spine density of mature and young neurons was not affected. Western blot analysis showed a progressive increase in the protein levels of glucocorticoid receptors (GRs) in the cultured primary hippocampal neurons during neuronal maturation. CONCLUSION: These data suggest that mature neurons are likely more vulnerable to chronic exposure to CORT; this may be due to their higher expression of GRs when compared to younger DG neurons.

BACE1 in the retina: a sensitive biomarker for monitoring early pathological changes in Alzheimer's disease.

Because of a lack of sensitive biomarkers, the diagnosis of Alzheimer's disease (AD) cannot be made prior to symptom manifestation. Therefore, it is crucial to identify novel biomarkers for the presymptomatic diagnosis of AD. While brain lesions are a major feature of AD, retinal pathological changes also occur in patients. In this study, we investigated the temporal changes in ß-site APP-cleaving enzyme 1 (BACE1) expression in the retina and brain to determine whether it could serve as a suitable biomarker for early monitoring of AD. APP/PS-1 transgenic mice, 3, 6 and 8 months of age, were used as an experimental group, and age-matched C57/BL6 wild-type mice served as the control group. In the Morris water maze test, there were no significant differences in escape latency or in the number of crossings in the target area among mice of different ages. Compared with wild-type mice, no changes in learning or memory abilities were detected in transgenic mice at 3 months of age. However, compared with wild-type mice, the escape latency was significantly increased in transgenic mice at 6 months, starting on day 3, and at 8 months, starting on day 2, during Morris water maze training. In addition, the number of crossings of the target area was significantly decreased in transgenic mice. The learning and memory abilities of transgenic mice were further worsened at 8 months of age. Immunohistochemical staining revealed no BACE1 plaques in wild-type mice at 3, 6 or 8 months or in transgenic mice at 3 months, but they were clearly found in the entorhinal cortex, hippocampus and prefrontal cortex of transgenic mice at 6 and 8 months. BACE1 expression was not detected in the retina of wild-type mice at 3 months, but weak BACE1 expression was detected in the ganglion cell layer, inner plexiform layer and outer plexiform layer at 6 and 8 months. In transgenic mice, BACE1 expression in the ganglion cell layer was increased at 3 months, and BACE1 expression in the ganglion cell layer, inner plexiform layer and outer plexiform layer was significantly increased at 6 and 8 months, compared with age-matched wild-type mice. Taken together, these results indicate that changes in BACE1 expression appear earlier in the retina than in the brain and precede behavioral deficits. Our findings suggest that abnormal expression of BACE1 in the retina is an early pathological change in APP/PS-1 transgenic mice, and that BACE1 might have potential as a biomarker for the early diagnosis of AD in humans.

Age of rats seriously affects the degree of retinal damage induced by acute high intraocular pressure.

PURPOSE: To investigate whether retinal impairment was affected by age of rats in acute glaucoma model. METHODS: Young adult and aged rats were randomly divided into normal control, 45 mmHg, 60 mmHg and 90 mmHg groups. Intraocular pressures (IOP) of rats were acutely elevated to 45 mmHg, 60 mmHg and 90 mmHg, respectively. Neuron loss in ganglion cell layer (GCL) and activation of retinal macrolgia and microglia 3 days after high IOP treatment were detected by immunofluorescence and further quantitatively analyzed. RESULTS: Compared with normal control, significant loss of neurons at GCL of young adult retina wasn't detected until IOP treatment of 90 mmHg. In contrast, obvious loss of neurons at GCL of aged retina was detected at IOP of 45 mmHg (p = 0.002 for central; p = 0.001 for peripheral). The loss level of neurons of aged retina was significantly higher than that of young adult retina at different IOP treatments. Compared with the young adult retina, high IOP induced more significant increase at area percentage of microglia and microglia number in inner part of aged retina. Activation of microglia and macroglia was either in parallel to or earlier than neuron loss of GCL of aged and young adult retina. CONCLUSION: Our data suggest there exists an age-related susceptibility of rat retina to the increased IOP. Therefore, the effect of ages should be considered at glaucoma study of rat models.

Differential neuronal expression of receptor interacting protein 3 in rat retina: involvement in ischemic stress response.

BACKGROUND: Receptor-interacting protein 3 (RIP3), a member of RIP family proteins, has been shown to participate in programmed necrosis or necroptosis in cell biology studies. Evidence suggests that necroptosis may be a mode of neuronal death in the retina. RESULTS: In the present study we determined the expression of RIP3 in normal rat retina and its changes following acute high intraocular pressure (aHIOP). RIP3 immunoreactivity (IR) was largely present in the inner retinal layers, localized to subsets of cells expressing neuron-specific nuclear antigen (NeuN), parvalbumin and calbindin in the ganglion cell layer (GCL) and inner nuclear layer (INL). No double labeling was detected for RIP3 with PKC-α or rhodopsin. RIP3 immunoreactivity was increased in the GCL at 6 hr and 12 hr, but reduced at 24 hr in the retina, without apparent alteration in laminar or cellular distribution pattern. Western blot analysis confirmed the above time-dependent alteration in RIP3 protein expression. RIP3 expressing cells frequently co-localized with propidium iodide (PI). A few co-localized cells were observed between RIP3 and Bax or cleaved caspase-3 in the GCL in 12 hr following aHIOP. CONCLUSIONS: The results indicate that RIP3 is expressed differentially in retinal neurons in adult rats, including subsets of ganglion cells, amacrine and horizontal cells. RIP3 protein levels are elevated rapidly following aHIOP. RIP3 labeling co-localized with PI, Bax or cleaved caspase-3 among cells in the ganglion cell layer following aHIOP, which suggest its involvement of RIP3 in neuronal responses to acute ischemic insults.

Surgery upregulates high mobility group box-1 and disrupts the blood-brain barrier causing cognitive dysfunction in aged rats.

AIM: Postoperative cognitive dysfunction (POCD) is a growing and largely underestimated problem without defined etiology. Herein, we sought to determine the relationship between cognitive decline, blood-brain barrier (BBB) permeability, and inflammation, namely high mobility group box-1 (HMGB1), after surgery in aged rats. METHODS: Aged rats were randomly assigned as surgery group (n = 45, splenectomy under general anesthesia), anesthesia (n = 45, 2% isoflurane for 2 h), and naïve control (n = 15). Markers of inflammation were measured in plasma and brain. Blood-brain barrier ultrastructure and permeability were measured by transmission electron microscope (TEM) and IgG immunohistochemistry. Cognitive function was assessed in a reversal learning version of the Morris water maze (MWM). RESULTS: Surgical trauma under general anesthesia caused distinct changes in systemic and central proinflammatory cytokines. Levels of HMGB1 and the receptor for advanced glycation end products (RAGE) were significantly upregulated in the hippocampus of operated animals. Immunohistochemistry and TEM showed BBB disruption induced by surgery and anesthesia. These molecular changes were associated with cognitive impairment in latency with the MWM up to postoperative day 3. CONCLUSIONS: HMGB1 and RAGE signaling appear pivotal mediators of surgery-induced cognitive decline and may contribute to the changes in BBB permeability after peripheral surgical trauma.

Hippocampal neurogenesis and dendritic plasticity support running-improved spatial learning and depression-like behaviour in stressed rats.

Exercise promotes hippocampal neurogenesis and dendritic plasticity while stress shows the opposite effects, suggesting a possible mechanism for exercise to counteract stress. Changes in hippocampal neurogenesis and dendritic modification occur simultaneously in rats with stress or exercise; however, it is unclear whether neurogenesis or dendritic remodeling has a greater impact on mediating the effect of exercise on stress since they have been separately examined. Here we examined hippocampal cell proliferation in runners treated with different doses (low: 30 mg/kg; moderate: 40 mg/kg; high: 50 mg/kg) of corticosterone (CORT) for 14 days. Water maze task and forced swim tests were applied to assess hippocampal-dependent learning and depression-like behaviour respectively the day after the treatment. Repeated CORT treatment resulted in a graded increase in depression-like behaviour and impaired spatial learning that is associated with decreased hippocampal cell proliferation and BDNF levels. Running reversed these effects in rats treated with low or moderate, but not high doses of CORT. Using 40 mg/kg CORT-treated rats, we further studied the role of neurogenesis and dendritic remodeling in mediating the effects of exercise on stress. Co-labelling with BrdU (thymidine analog) /doublecortin (immature neuronal marker) showed that running increased neuronal differentiation in vehicle- and CORT-treated rats. Running also increased dendritic length and spine density in CA3 pyramidal neurons in 40 mg/kg CORT-treated rats. Ablation of neurogenesis with Ara-c infusion diminished the effect of running on restoring spatial learning and decreasing depression-like behaviour in 40 mg/kg CORT-treated animals in spite of dendritic and spine enhancement. but not normal runners with enhanced dendritic length. The results indicate that both restored hippocampal neurogenesis and dendritic remodelling within the hippocampus are essential for running to counteract stress.

Preparation of novel anti-ski monoclonal antibodies.

Ski is an avian sarcoma virus oncogene homolog best known for inhibiting TGF beta signaling through its association with the SMAD proteins. Anti-Ski antibodies (MAbs) of high titer were prepared by immunizing BALB/c mice with multifocal intradermal injections and fusing high titer antibody producing spleen cells with myeloma cells of SP2/0 origin. Three MAbs were selected for further characterization as classes and subclasses. Antibodies were produced by these three clones with high affinities ranging from 10(9) to 10(11)/m. These clones were found to be of the immunoglobulin IgG1 and IgG2b subclass with kappa light chain. They could recognize Ski as determined by Western blot analysis. The produced MAbs will be a useful tool for further investigation of Ski functions in organisms.

Comparative proteome analysis of 3T3-L1 adipocyte differentiation using iTRAQ-coupled 2D LC-MS/MS.

Adipose tissue is critical in obesity and type II diabetes. Blocking of adipocyte differentiation is one of the anti-obesity strategies targeting on strong rise in fat storage and secretion of adipokine(s). However, the molecular basis of adipocyte differentiation and its regulation remains obscure. Therefore, we exposed 3T3-L1 cell line to appropriate hormonal inducers as adipocyte differentiation model. Using iTRAQ-coupled 2D LC-MS/MS, a successfully exploited high-throughput proteomic technology, we nearly quantitated 1,000 protein species and found 106 significantly altered proteins during adipocyte differentiation. The great majority of differentially expressed proteins were related to metabolism enzymes, structural molecules, and proteins involved in signal transduction. In addition to previously reported differentially expressed molecules, more than 20 altered proteins previously unknown to be involved with adipogenic process were firstly revealed (e.g., HEXB, DPP7, PTTG1IP, PRDX5, EPDR1, SPNB2, STEAP3, TPP1, etc.). The partially differential proteins were verified by Western blot and/or real-time PCR analysis. Furthermore, the association of PCX and VDAC2, two altered proteins, with adipocyte conversion was analyzed using siRNA method, and the results showed that they could contribute considerably to adipogenesis. In conclusion, our data provide valuable information for further understanding of adipogenesis.

Differential changes of local blood supply in rat retinae are involved in the selective loss of retinal ganglion cells following the acute high intraocular pressure.

PURPOSE: To detect changes in local blood supply to central, middle, and peripheral retina following acute high intraocular pressure and to investigate the effects of changes in local retinal blood supply on the selective loss of retinal ganglion cells. METHODS: The acute high intraocular pressure model of Sprague-Dawley rats was established by increasing the anterior chamber pressure to 110 mmHg via a normal saline perfusion system. Blood supply to the central, middle, and peripheral retina at 3, 6, and 12 h, and 1, 3, 7, and 14 d following induction of acute high intraocular pressure was detected by using gelatin-ink perfusion and fluorescent microsphere injection. Retinal ganglion cell loss following acute high intraocular pressure was detected by fluorogold retrograde labeling. Finally, the relationship between changes in local retinal blood supply and loss of retinal ganglion cells was investigated. RESULTS: The increased ratio of blood supply of peripheral retina was less than that of the central and middle retina at 3 h to 14 d following acute high intraocular pressure. The percent of retinal ganglion cell loss in the peripheral retina was clearly greater than that in the central and middle retina during the first 3 d following induction of acute high intraocular pressure (p < 0.05). Using either the gelatin-ink infusion method or the microsphere injection method (p < 0.05), a significantly negative correlation between the percent of retinal ganglion cell loss and the corresponding increased local blood supply after induction of acute high intraocular pressure (r = -0.57 or -0.72) was found. Moreover, a significant negative correlation in the peripheral retina (r = -0.80 or -0.91; p < 0.05) was observed. CONCLUSION: A disparity exists between changes in local blood supply to the central and middle retina, and the peripheral retina following induction of acute high intraocular pressure in rats. This inequality of changes in local blood supply in rat retina is related to the selective loss of retinal ganglion cells.

[A novel mutation of CYP1B1 gene in primary congenital glaucoma].

OBJECTIVE: To investigate the distribution of the CYP1B1 (Cytochrome P450, family 1, subfamily B, polypeptide 1) gene mutations in primary congenital glaucoma (PCG) in Hunan Province. METHODS: Case-control study. Thirteen cases of PCG from different districts of Hunan province were collected in this study. Direct sequencing was used to evaluate the coding and the promoter regions of the CYP1B1 gene in PCG patients. RESULTS: A novel pathogenic mutation (c.C319G, L107V) was identified in a PCG patient in our study and it was a missense mutation in exon 2. Additionally, four single nucleotide polymorphisms(SNPs) were found in PCG patients, including R48G, A119S, V432L and D449D. CONCLUSION: A novel CYP1B1 gene mutation (L107V) may be the cause for primary congenital glaucoma in Hunan Province.