Mesenchymal stromal cell-derived small extracellular vesicles promote neurological recovery and brain remodeling after distal middle cerebral artery occlusion in aged rats

Small extracellular vesicles (sEVs) obtained from mesenchymal stromal cells (MSCs) promote neurological recovery after middle cerebral artery occlusion (MCAO) in young rodents. Ischemic stroke mainly affects aged humans. MSC-sEV effects on stroke recovery in aged rodents had not been assessed. In a head-to-head comparison, we exposed young (4–5 months) and aged (19–20 months) male Sprague–Dawley rats to permanent distal MCAO. At 24 h, 3 and 7 days post-stroke, vehicle or MSC-sEVs (2 × 106 or 2 × 107 MSC equivalents/kg) were intravenously administered. Neurological deficits, ischemic injury, brain inflammatory responses, post-ischemic angiogenesis, and endogenous neurogenesis were evaluated over 28 days. Post-MCAO, aged vehicle-treated rats exhibited more severe motor-coordination deficits evaluated by rotating pole and cylinder tests and larger brain infarcts than young vehicle-treated rats. Although infarct volume was not influenced by MSC-sEVs, sEVs at both doses effectively reduced motor-coordination deficits in young and aged rats. Brain macrophage infiltrates in periinfarct tissue, which were evaluated as marker of a recovery-aversive inflammatory environment, were significantly stronger in aged than young vehicle-treated rats. sEVs reduced brain macrophage infiltrates in aged, but not young rats. The tolerogenic shift in immune balance paved the way for structural brain tissue remodeling. Hence, sEVs at both doses increased periinfarct angiogenesis evaluated by CD31/BrdU immunohistochemistry in young and aged rats, and low-dose sEVs increased neurogenesis in the subventricular zone examined by DCX/BrdU immunohistochemistry. Our study provides robust evidence that MSC-sEVs promote functional neurological recovery and brain tissue remodeling in aged rats post-stroke. This study encourages further proof-of-concept studies in clinic-relevant stroke settings.

Neurogenic Plasticity in a Serotonin Transporter Knockout Model: Effects of Maternal and Offspring Genotype

<p>Major depressive disorder (MDD) is debilitating mental disorder that is increasing in prevalence. Many theories have tried to explain the aetiology of depression including the classic monoamine deficiency hypothesis and the newer neurogenic hypothesis. The finding that selective serotonin transporter inhibitors (SSRIs) work by increasing extracellular serotonin levels in the brain and have antidepressant effects has formed the basis of the most widely accepted theory of depression, the monoamine hypothesis. However, a genetic reduction in human and animal serotonin reuptake transporters, which also increases extracellular serotonin, is associated with depressive symptomology. This paradox is not explained by the monoamine hypothesis. The key difference between these two scenarios is that genetically induced increases in serotonin occur from development onward, while SSRIs increase serotonin only in adulthood. Furthermore, SSRIs typically take several weeks to confer a therapeutic effect. This finding has led to the hypothesis that, rather than acute monoamine-increasing effects, it is the downstream effects of such increases on neurogenesis and neural plasticity which confer antidepressant effects. To further elucidate the neurobiology of depression, this study sought to examine the effects of genetically increasing serotonin on early postnatal neurogenesis in a serotonin knockout rat model using BrdU immunohistochemistry. We examined both the offspring and maternal genotype effects. We found that SERT-/- offspring had the highest levels of neurogenesis compared with SERT+/- and SERT+/+ at postnatal day 7. In addition we found a maternal genotype effect with SERT+/+ offspring born and reared by SERT+/- mothers having lower neurogenesis compared to SERT+/+ offspring from SERT+/+ mothers. The potential effects of maternal caregiving, neuroplasticity in altered mood and stress responses and the role of 5-HT receptors are discussed.</p>

Neurogenic Plasticity in a Serotonin Transporter Knockout Model: Effects of Maternal and Offspring Genotype

<p>Major depressive disorder (MDD) is debilitating mental disorder that is increasing in prevalence. Many theories have tried to explain the aetiology of depression including the classic monoamine deficiency hypothesis and the newer neurogenic hypothesis. The finding that selective serotonin transporter inhibitors (SSRIs) work by increasing extracellular serotonin levels in the brain and have antidepressant effects has formed the basis of the most widely accepted theory of depression, the monoamine hypothesis. However, a genetic reduction in human and animal serotonin reuptake transporters, which also increases extracellular serotonin, is associated with depressive symptomology. This paradox is not explained by the monoamine hypothesis. The key difference between these two scenarios is that genetically induced increases in serotonin occur from development onward, while SSRIs increase serotonin only in adulthood. Furthermore, SSRIs typically take several weeks to confer a therapeutic effect. This finding has led to the hypothesis that, rather than acute monoamine-increasing effects, it is the downstream effects of such increases on neurogenesis and neural plasticity which confer antidepressant effects. To further elucidate the neurobiology of depression, this study sought to examine the effects of genetically increasing serotonin on early postnatal neurogenesis in a serotonin knockout rat model using BrdU immunohistochemistry. We examined both the offspring and maternal genotype effects. We found that SERT-/- offspring had the highest levels of neurogenesis compared with SERT+/- and SERT+/+ at postnatal day 7. In addition we found a maternal genotype effect with SERT+/+ offspring born and reared by SERT+/- mothers having lower neurogenesis compared to SERT+/+ offspring from SERT+/+ mothers. The potential effects of maternal caregiving, neuroplasticity in altered mood and stress responses and the role of 5-HT receptors are discussed.</p>

Bromodeoxyuridine (BrdU) marker for proliferating cells in living tissues: Detection in adult avian brain

Knowing how to perform cell proliferation assays is an undeniably essential skill to detect the effect of external or internal stimuli on cell genesis. Immunohistochemical staining using Bromodeoxyuridine (BrdU), enables quantification of proliferative fraction of cells in sectioned tissues. BrdU, an artificial nucleoside that substitutes for thymidine. It is currently quite popular research tool of choice for new cell genesis studies. It incorporates into the DNA during S-phase of the cell cycle that’s why used for birth dating and observing cell proliferation. BrdU immunohistochemistry is currently the most used technique for studying adult cell genesis in situ. In this article we provide a step-by step protocol for immunodetection of BrdU by light microscopy using avian species (Ploceus philippinus); starting from BrdU administration to the final analysis.

Evaluation of gamma ray-induced gastrointestinal tract morphological and proliferative activity changes in rhesus monkeys

To provide support for future pharmacology and preclinical studies, we have established a stable nonhuman primate animal model to demonstrate the histopathological changes in the gastrointestinal tract following gamma ray irradiation. In this study, 12 healthy rhesus monkeys were divided into 2 groups (control and radiation groups). Animals in the radiation group were exposed to gamma rays (cobalt 60 source) at a dose level of 6.5 Gy total body irradiation bilaterally (i.e. 3.25 Gy on each side). Control animals were sham exposed using identical procedures. After a 5-day in-life observation period, gastrointestinal tract tissues (esophagus, stomach, duodenum, jejunum, ileum, colon, and rectum) were collected and fixed in 10% neutral-buffered formalin for subsequent hematoxylin and eosin and 5-bromo-2-deoxyuridine (BrdU) immunohistochemistry processing. The results showed that the esophagus was undergoing degeneration without obvious inflammatory changes, while the stomach and duodenum exhibited both degeneration and inflammation. From the jejunum to the rectum, late-stage inflammation with glandular regeneration, as well as a high-level BrdU labeling index, was present.

Involvement of CaMKIV in neurogenic effect with chronic fluoxetine treatment

Calcium-calmodulin dependent protein kinase IV (CaMKIV) is a protein kinase that has been suggested to participate in fluoxetine (FLX)-induced phosphorylation of cyclic AMP-response element binding protein (CREB). CREB is a key transcription factor in adult neurogenesis. The present study aimed at evaluating whether CaMKIV is involved in adult hippocampal neurogenesis with FLX treatment. Effects of chronic FLX on hippocampal cell proliferation, survival and phenotypes were assessed using bromodeoxyuridine (BrdU) immunohistochemistry or BrdU/neuronal nuclei (NeuN)/S100β immunofluorescence staining in wild-type (WT) and CaMKIV knockout (KO) mice. Expression and phosphorylation of CaMKIV and CREB were assessed using RT–PCR and Western blotting. The behavioural action with FLX was assessed in the novelty suppressed feeding test (NSF), which is considered neurogenesis-dependent. CaMKIV KO mice have reduced cell proliferation, but not survival in the dentate gyrus of hippocampus with chronic treatment of FLX when compared to wild littermates. Phenotype analysis showed that most newborn cells matured into neurons. Phosphorylation of CaMKIV was up-regulated in WT mice and phosphorylation of CREB was impaired in CaMKIV KO mice after FLX treatment. The behavioural effects of FLX in NSF were similar in both types. These data suggest that CaMKIV is involved in some aspects of FLX-promoting hippocampal neurogenesis.

240. Reduced endometrial angiogenesis during early pregnancy in relaxin-deficient mice

Relaxin is a peptide hormone with important roles in the reproductive tract, including the growth and remodelling of endometrial vasculature. It has been shown to stimulate VEGF secretion from human endometrial stromal cells in vitro and increases endometrial vascularisation in ovariectomised steroid-primed primates in vivo. We have used mouse models to show that oestrogen and progesterone stimulate angiogenesis (new blood vessel growth) within the endometrium. Endometrial angiogenesis also occurs in the early stages of mouse pregnancy, which coincides with an increase in circulating progesterone. To date, no studies have investigated the effects of relaxin on endometrial angiogenesis in early pregnancy. Our aim was to test the hypothesis that endometrial angiogenesis would be reduced in relaxin-deficient mice (Rln−/−) in comparison to their wildtype (Rln+/+) counterparts. Uterine tissues were collected from Rln−/− and Rln+/+ mice on days 1 to 4 of pregnancy, before implantation. All mice were treated with BrdU before dissection to allow the number of blood vessel profiles containing proliferating endothelial cells (PVPs) to be quantified by double CD31/BrdU immunohistochemistry. Consistent with published studies, PVPs were first observed on days 3 and 4 of pregnancy. However, the percentage of PVPs was reduced in Rln−/− mice compared with Rln+/+ mice (Day 3: median = 4.4% v. 19.6%, Day 4: 9.6% v. 22.2%). We subsequently identified relaxin and relaxin receptors in the mouse endometrium in early pregnancy. Our data suggest that locally synthesised relaxin acts in synergy with progesterone to initiate endometrial angiogenesis in early pregnancy.

Light and Electron Microscopic Immunohistochemical Detection of Bromodeoxyuridine-labeled Cells in the Brain: Different Fixation and Processing Protocols

Bromodeoxyuridine (BrdU) immunohistochemistry is the method of choice for labeling newly generated cells in the brain. Most BrdU studies utilize paraformaldehyde-fixed brain tissue because of its compatibility with both BrdU and other immunohistochemical methods. However, stronger fixation is required for electron microscopic studies, and unfixed tissue is needed for biochemical and molecular studies. Because there are no systematic studies comparing the effects of different fixatives on BrdU immunohistochemistry in brain tissue, we compared BrdU immunohistochemical methods in brain tissue fixed with 4% paraformaldehyde, a mixed glutaraldehyde–paraformaldehyde fixative for electron microscopy, and unfixed tissue from brains perfused only with buffer and flash frozen. After optimizing immunostaining protocols, qualitative assessments of light microscopic diaminobenzidine labeling and of double-label immunofluorescence with confocal microscopy demonstrated excellent BrdU labeling in each of the three groups. Quantitative stereological assessment of the number of BrdU-labeled cells in rat dentate gyrus showed no significant difference in the number of labeled cells detected with each perfusion protocol. Additionally, we developed a protocol to visualize BrdU-labeled cells in the electron microscope with adequate preservation of fine structure in both rat and monkey brain.