Intramammary Labeling of Epithelial Cell Division.

Thymidine analogs such as ethynyl deoxyuridine (EdU) or bromodeoxyuridine (BrdU) can be used to label mitosis of mammary epithelial cells (MEC) and to quantify their proliferation. However, labeling cells in larger animals requires considerable amounts of chemical that can be costly and hazardous. We developed a strategy to infuse EdU into the mammary glands of ewes to directly label mitotic MEC. First, each udder half of nulliparous ewes (n = 2) received an intramammary infusion of one of four different concentrations of EdU (0, 0.1, 1.0 or 10 mM) which was compared to BrdU IV (5 mg/kg) 24 h later. Tissues were analyzed by immunofluorescent histochemistry to detect EdU, BrdU, and total MEC. Of the EdU doses tested, 10 mM EdU yielded the greatest labeling index, while a proportion of MEC were labeled by both EdU and BrdU. We next sought to establish whether intramammary labeling could detect the induction of mitosis after exposure to exogenous estrogen and progesterone (E + P). We first infused EdU (10 mM) into the right udder half of ewes (n = 6) at t 0, followed by thymidine (100 mM) 24 h later to prevent further labeling. Three ewes were then administered E + P for 5 d, while n = 3 ewes served as controls. On d 5, EdU was infused into the left udder half of all mammary glands alongside BrdU IV (5 mg/kg). By the time of necropsy 24 h later an average MEC labeling index of 2.9% resulted from EdU delivered at t 0. In the left half of the udder on d 5, CON glands had a final EdU labeling index of 3.4% while glands exposed to E + P had a labeling index of 4.6% (p = 0.05). The corresponding degree of labeling with BrdU was 5.6% in CON glands, and 12% following E + P (p < 0.001). Our findings reveal that intramammary labeling is an efficient and cost-effective method for single- and dual-labeling of cell division in the mammary glands.

Cross-kingdom interactions and functional patterns of active microbiota matter in governing deadwood decay.

Microbial community members are the primary microbial colonizers and active decomposers of deadwood. This study placed sterilized standardized beech and spruce sapwood specimens on the forest ground of 8 beech- and 8 spruce-dominated forest sites. After 370 days, specimens were assessed for mass loss, nitrogen (N) content and 15N isotopic signature, hydrolytic and lignin-modifying enzyme activities. Each specimen was incubated with bromodeoxyuridine (BrdU) to label metabolically active fungal and bacterial community members, which were assessed using amplicon sequencing. Fungal saprotrophs colonized the deadwood accompanied by a distinct bacterial community that was capable of cellulose degradation, aromatic depolymerization, and N2 fixation. The latter were governed by the genus Sphingomonas, which was co-present with the majority of saprotrophic fungi regardless of whether beech or spruce specimens were decayed. Moreover, the richness of the diazotrophic Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium group was significantly correlated with mass loss, N content and 15N isotopic signature. By contrast, presence of obligate predator Bdellovibrio spp. shifted bacterial community composition and were linked to decreased beech deadwood decay rates. Our study provides the first account of the composition and function of metabolically active wood-colonizing bacterial and fungal communities, highlighting cross-kingdom interactions during the early and intermediate stages of wood decay.

The influence of increased water temperature on the duration of spermatogenesis in a neotropical fish, Astyanax altiparanae (Characiformes, Characidae).

In view of the established climate change scenario and the consequent changes in global temperature, it is essential to study its effects on animal spermatogenesis. Therefore, the aim of this study was to verify the duration of spermatogenesis at different temperatures. For this purpose, 96 male and adult specimens of Astyanax altiparanae were kept in a closed circulation system with water temperature stabilized at 27 °C and 32 °C. Subsequently, the specimens received pulses of BrdU (bromodeoxyuridine) at a concentration of 100 mg/kg/day for 2 consecutive days, and the samples were collected daily for a period of 15 days. Their testes were removed, fixed, processed in historesin, and sectioned in 3 µm, submitted to hematoxylin/eosin staining and to bromodeoxyuridine immunodetection. Partial results of the optimum temperature experiments allowed the classification of A. altiparanae spermatogenic cells in Aund, Adiff, and type B spermatogonia, spermatocytes, spermatids, and spermatozoa. The duration of spermatogenesis was determined as approximately 6 days for animals at a temperature of 27 °C and 1 day for animals at 32 °C. The elevated temperature was also responsible for increasing cell proliferation, resulting in an increase in the number of spermatocytes, spermatids, spermatozoa, and cell death (cell pyknotic). The duration of spermatogenesis in A. altiparanae was directly affected by the elevated water temperature, causing a reduction in the estimated time of spermatogenesis.

Adult neuronal regeneration in the telencephalon of the killifish Aphaniops hormuzensis.

The potential of central nervous system regeneration was evaluated for the first time in the injured brain of the old world killifish Aphaniops hormuzensis. The histomorphological organization in the regeneration procedure was evaluated using the hematoxylin and eosin (H&E) staining and the bromodeoxyuridine (BrdU) immunohistochemistry technique. The histological tissue sections were sampled daily for 10 days. Based on the H&E staining, a large gliosis reaction was detected along with vacuolization and telencephalon deformation on 1-day post-lesion (dpl). The vacuolated zone declined fast and the telencephalon hemisphere recovered on 3 dpl. The symptoms of injured telencephalon nervous tissue were resolved within 7 dpl in both genders. In the BrdU test of the control group, BrdU-labeled cells were observed in the ventricular zone (VZ), pallium (Pa), and lateral pallium (LPa). On 1 dpl, the BrdU+ cells accumulated in the VZ, Pa, and LPa (located near the injury area). From 3 dpl onwards, the BrdU+ cells were reduced in the telencephalic VZ, Pa, and LPa. Based on the BrdU+ results, the adult brain in A. hormuzensis possesses a remarkable capacity for neuronal regeneration. By taking into account the high neural regeneration potency of A. hormuzensis and its relatively short lifespan, it could be concluded that besides the currently known models, the members of aphaniid fishes could probably be valuable animals to study the regeneration phenomenon in the vertebrates.

Factors effecting the induction of rat forestomach hyperplasia induced by Swedish oral smokeless tobacco (snus).

Long term exposure to oral smokeless tobacco may induce lesions in the oral cavity characterized by a hyperplastic epithelium. The possible role of nicotine and the physical properties of oral tobacco for developing these lesions, as well as of dysplasia and neoplasia is unclear. Low nitrosamine Swedish snus as well as non-genotoxic butylated hydroxyanisole induces increased cellular proliferation in the rat forestomach epithelia. Using this model, we report here on the effects of nicotine, pH, and particle size. Snus with different properties had no impact on oxidative stress as determined by 8-oxo-7,8-dihydro-2'-deoxyguanosine, or on interleukin IL-1b. Whereas BHA boosted IL-6, probably due to the presence of nicotine. there was no significant enhancement of cell divisions with increasing particle size, although in individual samples the variations in proliferation rates increased greatly with increasing particle size. Conforming to human experience, the enhanced cell proliferation caused by snus was found to be completely reversible. A cacao bean extract had a protective action similar to that previously found for blueberries. The main cause of the observed tobacco induced cell proliferation could be mechanical irritation, possibly in combination with nicotine, whereas within the studied range, pH did not affect the rate of cell division.

Changes in the Distribution of Cell Contacts and Mitotic Cycle Disturbances in Cells of the Allograft of Rat Embryonic Neocortex.

Morphological changes in the allograft of rat anterior cerebral vesicle at the early stages after transplantation into the peripheral nerve of an adult rat were studied by immunohistochemical methods. Immunohistochemical reaction to bromodeoxyuridine showed that the delay of mitotic division in neural stem/progenitor cells in the grafts occurred during S/G2 stage. In transplants of rat embryonic neocortex (E13), changes in the cell cycle of neural stem/progenitor cells in 3 h after transplantation into the nerve correlated with abnormal distribution of adherens junctions and interkinetic nuclear migration.

Cells of NG2 lineage increase in glomeruli of mice following podocyte depletion.

Under certain circumstances, podocytes can be partially replaced following their loss in disease. The inability of podocytes to proliferate suggests that replacement derives from other cell types. Because neural/glial antigen 2 (NG2)-expressing cells can serve as progenitors in other organs and because herein we showed increased NG2 staining in podocytes following their loss in experimental focal segmental glomerulosclerosis, we used lineage tracing in NG2-CreER tdTomato mice to test the hypothesis that partial podocyte replacement might derive from this cell population. The percentage of glomeruli with red fluorescence protein (RFP)-labeled NG2 cells increased following podocyte depletion, which was augmented by enalapril. However, BrdU was not detected in RFP-labeled cells, consistent with the migration of these cells to the glomerulus. Within glomeruli, RFP-labeled cells did not coexpress podocyte proteins (p57, synaptopodin, nephrin, or podocin) but did coexpress markers for mesangial (α8 integrin, PDGFß receptor) and parietal epithelial cells (PAX8, src-suppressed C-kinase substrate). These results suggest that following podocyte depletion, cells of NG2 lineage do not serve as adult podocyte progenitors but have the ability to transdifferentiate to mesangial and parietal epithelial cell fates.

Main Olfactory and Vomeronasal Epithelium Are Differently Affected in Niemann-Pick Disease Type C1.

INTRODUCTION: Olfactory impairment is one of the earliest symptoms in neurodegenerative disorders that has also been documented in Niemann-Pick disease type C1 (NPC1). NPC1 is a very rare, neurovisceral lipid storage disorder, characterized by a deficiency of Npc1 gene function that leads to progressive neurodegeneration. Here, we compared the pathologic effect of defective Npc1 gene on the vomeronasal neuroepithelium (VNE) with that of the olfactory epithelium (OE) in an NPC1 mouse model. METHODS: Proliferation in the VNE and OE was assessed by applying a bromodeoxyuridine (BrdU) protocol. We further compared the immunoreactivities of anti-olfactory marker protein (OMP), and the lysosomal marker cathepsin-D in both epithelia. To investigate if degenerative effects of both olfactory systems can be prevented or reversed, some animals were treated with a combination of miglustat/allopregnanolone/2-hydroxypropyl-cyclodextrin (HPßCD), or a monotherapy with HPßCD alone. RESULTS: Using BrdU to label dividing cells of the VNE, we detected a proliferation increase of 215% ± 12% in Npc1-/- mice, and 270% ± 10% in combination- treated Npc1-/- animals. The monotherapy with HPßCD led to an increase of 261% ± 10.5% compared to sham-treated Npc1-/- mice. Similar to the OE, we assessed the high regenerative potential of vomeronasal progenitor cells. OMP reactivity in the VNE of Npc1-/- mice was not affected, in contrast to that observed in the OE. Concomitantly, cathepsin-D reactivity in the VNE was virtually absent. Conclusion: Vomeronasal receptor neurons are less susceptible against NPC1 pathology than olfactory receptor neurons. Compared to control mice, however, the VNE of Npc1-/- mice displays an increased neuroregenerative potential, indicating compensatory cell renewal.

Quiescent adult stem cells in murine teeth are regulated by Shh signaling.

The mechanisms regulating the maintenance of quiescent adult stem cells in teeth remain to be fully elucidated. Our aim is to clarify the relationship between BrdU label-retaining cells (LRCs) and sonic hedgehog (Shh) signaling in murine teeth. After prenatal BrdU labeling, mouse pups were analyzed during postnatal day 1 (P1) to week 5 (P5W). Paraffin sections were processed for immunohistochemistry for BrdU, Sox2, Gli1, Shh, Patched1 (Ptch1) and Ki67 and for in situ hybridization for Shh and Ptch1. Dense LRCs, Gli1-(+) cells and Ptch1-(+) cells were co-localized in the outer enamel epithelium of the apical bud and apical dental papilla of incisors. In developing molars, dense LRCs were numerous at P1 but then decreased in number over the course of odontogenesis and were maintained in the center of pulp tissue. Gli1-(+) cells were maintained in the pulp horn during the examined stages, while they increased in number and were maintained in the center of pulp tissue during P2-5W. Ptch1-(+) cells were localized in the pulp horn at P1 and increased in number in the center of the pulp after P3W. Shh mRNA was first expressed in the enamel epithelium and then shifted to odontoblasts and other pulp cells. Shh protein was distributed in the epithelial and mesenchymal tissues of incisors and molars. These findings suggest that quiescent dental stem cells are regulated by Shh signaling, and that Shh signaling plays a crucial role in the differentiation and integrity of odontoblasts during epithelial-mesenchymal interactions and dentinogenesis.

Stretched cell cycle model for proliferating lymphocytes.

Stochastic variation in cell cycle time is a consistent feature of otherwise similar cells within a growing population. Classic studies concluded that the bulk of the variation occurs in the G1 phase, and many mathematical models assume a constant time for traversing the S/G2/M phases. By direct observation of transgenic fluorescent fusion proteins that report the onset of S phase, we establish that dividing B and T lymphocytes spend a near-fixed proportion of total division time in S/G2/M phases, and this proportion is correlated between sibling cells. This result is inconsistent with models that assume independent times for consecutive phases. Instead, we propose a stretching model for dividing lymphocytes where all parts of the cell cycle are proportional to total division time. Data fitting based on a stretched cell cycle model can significantly improve estimates of cell cycle parameters drawn from DNA labeling data used to monitor immune cell dynamics.

Increased Regenerative Capacity of the Olfactory Epithelium in Niemann-Pick Disease Type C1.

Niemann-Pick disease type C1 (NPC1) is a fatal neurovisceral lysosomal lipid storage disorder. The mutation of the NPC1 protein affects the homeostasis and transport of cholesterol and glycosphingolipids from late endosomes/lysosomes to the endoplasmic reticulum resulting in progressive neurodegeneration. Since olfactory impairment is one of the earliest symptoms in many neurodegenerative disorders, we focused on alterations of the olfactory epithelium in an NPC1 mouse model. Previous findings revealed severe morphological and immunohistochemical alterations in the olfactory system of NPC1-/- mutant mice compared with healthy controls (NPC1+/+). Based on immunohistochemical evaluation of the olfactory epithelium, we analyzed the impact of neurodegeneration in the olfactory epithelium of NPC1-/- mice and observed considerable loss of mature olfactory receptor neurons as well as an increased number of proliferating and apoptotic cells. Additionally, after administration of two different therapy approaches using either a combination of miglustat, 2-hydroxypropyl-ß-cyclodextrin (HPßCD) and allopregnanolone or a monotherapy with HPßCD, we recorded a remarkable reduction of morphological damages in NPC1-/- mice and an up to four-fold increase of proliferating cells within the olfactory epithelium. Numbers of mature olfactory receptor neurons doubled after both therapy approaches. Interestingly, we also observed therapy-induced alterations in treated NPC1+/+ controls. Thus, olfactory testing may provide useful information to monitor pharmacologic treatment approaches in human NPC1.

Sex and strategy use matters for pattern separation, adult neurogenesis, and immediate early gene expression in the hippocampus.

Adult neurogenesis in the dentate gyrus (DG) plays a crucial role for pattern separation, and there are sex differences in the regulation of neurogenesis. Although sex differences, favoring males, in spatial navigation have been reported, it is not known whether there are sex differences in pattern separation. The current study was designed to determine whether there are sex differences in the ability for separating similar or distinct patterns, learning strategy choice, adult neurogenesis, and immediate early gene (IEG) expression in the DG in response to pattern separation training. Male and female Sprague-Dawley rats received a single injection of the DNA synthesis marker, bromodeoxyuridine (BrdU), and were tested for the ability of separating spatial patterns in a spatial pattern separation version of delayed nonmatching to place task using the eight-arm radial arm maze. Twenty-seven days following BrdU injection, rats received a probe trial to determine whether they were idiothetic or spatial strategy users. We found that male spatial strategy users outperformed female spatial strategy users only when separating similar, but not distinct, patterns. Furthermore, male spatial strategy users had greater neurogenesis in response to pattern separation training than all other groups. Interestingly, neurogenesis was positively correlated with performance on similar pattern trials during pattern separation in female spatial strategy users but negatively correlated with performance in male idiothetic strategy users. These results suggest that the survival of new neurons may play an important positive role for pattern separation of similar patterns in females. Furthermore, we found sex and strategy differences in IEG expression in the CA1 and CA3 regions in response to pattern separation. These findings emphasize the importance of studying biological sex on hippocampal function and neural plasticity.

Aberrant localization of lamin B receptor (LBR) in cellular senescence in human cells.

5-Bromodeoxyuridine (BrdU), a thymidine analogue, induces cellular senescence in mammalian cells. BrdU induces cellular senescence probably through the regulation of chromatin because BrdU destabilizes or disrupts nucleosome positioning and decondenses heterochromatin. Since heterochromatin is tethered to the nuclear periphery through the interaction with the nuclear envelope proteins, we examined the localization of the several nuclear envelope proteins such as lamins, lamin-interacting proteins, nuclear pore complex proteins, and nuclear transport proteins in senescent cells. We have shown here that lamin B receptor (LBR) showed a change in localization in both BrdU-induced and replicative senescent cells.

Inhibition by blueberries (bilberries) and extract from milk thistle of rat forestomach hyperplasia induced by oral smokeless tobacco (Swedish snus).

The aim of this study was to identify palatable additives which have a significant protective action against soft tissue changes in the oral cavity caused by Swedish smokeless tobacco ("snus"), and that satisfy existing legal requirements. Although the cancer risk from snus is extremely low, long term use may result in highly undesirable keratotic lesions and associated epithelial abnormalities in the oral cavity. The rat forestomach, which is vulnerable to the irritative action of non-genotoxic compounds like butylated hydroxyanisole, propionic acid as well as snus, was chosen as an experimental model. Studied toxicological endpoints included histopathology and cellular proliferation based on DNA incorporation of bromodeoxyuridine. After 6 weeks' exposure, blueberries (bilberries) and an extract from the common milk thistle were found to exert a highly significant inhibition of cell proliferation induced by snus in the rat forestomach epithelium, indicating a potential protection with respect soft tissue changes in the human oral cavity.

Radiation-induced brain injury: low-hanging fruit for neuroregeneration.

Brain radiation is a fundamental tool in neurooncology to improve local tumor control, but it leads to profound and progressive impairments in cognitive function. Increased attention to quality of life in neurooncology has accelerated efforts to understand and ameliorate radiation-induced cognitive sequelae. Such progress has coincided with a new understanding of the role of CNS progenitor cell populations in normal cognition and in their potential utility for the treatment of neurological diseases. The irradiated brain exhibits a host of biochemical and cellular derangements, including loss of endogenous neurogenesis, demyelination, and ablation of endogenous oligodendrocyte progenitor cells. These changes, in combination with a state of chronic neuroinflammation, underlie impairments in memory, attention, executive function, and acquisition of motor and language skills. Animal models of radiation-induced brain injury have demonstrated a robust capacity of both neural stem cells and oligodendrocyte progenitor cells to restore cognitive function after brain irradiation, likely through a combination of cell replacement and trophic effects. Oligodendrocyte progenitor cells exhibit a remarkable capacity to migrate, integrate, and functionally remyelinate damaged white matter tracts in a variety of preclinical models. The authors here critically address the opportunities and challenges in translating regenerative cell therapies from rodents to humans. Although valiant attempts to translate neuroprotective therapies in recent decades have almost uniformly failed, the authors make the case that harnessing human radiation-induced brain injury as a scientific tool represents a unique opportunity to both successfully translate a neuroregenerative therapy and to acquire tools to facilitate future restorative therapies for human traumatic and degenerative diseases of the central nervous system.

Hominis Placenta facilitates hair re-growth by upregulating cellular proliferation and expression of fibroblast growth factor-7.

BACKGROUND: Hominis Placenta (HP) known as a restorative medicine in Traditional Chinese Medicine (TCM), has been widely applied in the clinics of Korea and China as an anti-aging agent to enhance the regeneration of tissue. This study was conducted to investigate whether topical treatment of HP promotes hair regrowth in the animal model. METHODS: The dorsal hairs of 8-week-old C57BL/6 mice were depilated to synchronize hair follicles to the anagen phase. HP was applied topically once a day for 15 days. Hair growth was evaluated visually and microscopically. The incorporation of bromodeoxyuridine (BrdU) and expression of proliferating cell nuclear antigen (PCNA), fibroblast growth factor-7 (FGF-7) in dorsal skin tissue was examined by immunohistochemical analysis. Reverse transcription polymerase chain reaction (RT-PCR) was used to measure the mRNA expression of FGF-7. RESULTS: HP exhibited potent hair growth-promoting activity in C57BL/6 mice. Gross examination indicated that HP markedly increased hair regrowth as well as hair density and diameter. Histologic analysis showed that HP treatment enhanced the anagen induction of hair follicles. Immunohistochemical analysis revealed that BrdU incorporation and the expressions of PCNA were increased by treatment of HP. HP treatment significantly increased the expression of FGF-7, which plays pivotal roles to maintain anagen phase both protein and mRNA levels. CONCLUSIONS: Taken together, our results indicate that HP has a potent hair growth-promoting activity; therefore, it may be a good candidate for the treatment of alopecia.

Argemone oil induces genotoxicity in mice.

CONTEXT: Argemone mexicana L. is native to Mexico and the plant extracts are used in traditional medicine in India and South American countries. Argemone oil (AO) is a common adulterant of mustard oil in India and causes serious pathophysiological consequences leading to outbreaks of epidemic dropsy among consumers. In vivo cytogenetic studies on the toxicological effects of AO and its component alkaloids are limited. OBJECTIVE: The present study was undertaken to evaluate the safety of AO by assessment of their in vivo genotoxic potential in bone marrow cells of mice. MATERIALS AND METHODS: AO mixed in corn oil in the proportions of 0.01, 0.1, and 1 ml AO/kg body weight in a total volume of 10 ml/kg body weight and a single undiluted dose of AO (10 ml/kg body weight) were administered intraperitoneally in separate groups of male Swiss Albino mice for 24 h. In addition, a single concentration of sanguinarine (SG) (50 mg/kg body weight) was also administered. Genotoxicity was evaluated by chromosome aberration (CA) and sister chromatid exchange (SCE) tests. Bromodeoxyuridine (BrdU) differential technique was used to study the effect on cell replication by the calculation of average generation time (AGT). RESULTS: The minimum effective concentrations that produced significant frequencies of CA and SCE were 0.1 and 0.01 ml/kg, respectively. AO and SG induced an insignificant increase of AGT indicating that they are non-cytotoxic in the concentrations tested. DISCUSSION AND CONCLUSION: Our results confirm that AO is genotoxic even at low concentrations and its usage should be checked.

Decreased cell proliferation and higher oxidative stress in fibroblasts from Down Syndrome fetuses. Preliminary study.

Down Syndrome is the most common chromosomal disease and is also known for its decreased incidence of solid tumors and its progeroid phenotype. Cellular and systemic oxidative stress has been considered as one of the Down Syndrome phenotype causes. We correlated, in a preliminary study, the fibroblast proliferation rate and different cell proliferation key regulators, like Rcan1 and the telomere length from Down Syndrome fetuses, with their oxidative stress profile and the Ribonucleic acid and protein expression of the main antioxidant enzymes together with their activity. Increased oxidized glutathione/glutathione ratio and high peroxide production were found in our cell model. These results correlated with a distorted antioxidant shield. The messenger RNA (SOD1) and protein levels of copper/zinc superoxide dismutase were increased together with a decreased mRNA expression and protein levels of glutathione peroxidase (GPx). As a consequence the [Cu/ZnSOD/(catalase+GPx)] activity ratio increases which explains the oxidative stress generated in the cell model. In addition, the expression of thioredoxin 1 and glutaredoxin 1 is decreased. The results obtained show a decreased antioxidant phenotype that correlates with increased levels of Regulator of calcineurin 1 and attrition of telomeres, both related to oxidative stress and cell cycle impairment. Our preliminary results may explain the proneness to a progeroid phenotype.

Tissue-type plasminogen activator is not necessary for platelet-derived growth factor-c activation.

Platelet-derived growth factors (PDGFs) are critical for development; their over-expression is associated with fibrogenesis. Full-length PDGF-C is secreted as an inactive dimer, requiring cleavage to allow receptor binding. Previous studies indicate that tissue-type plasminogen activator (tPA) is the specific protease that performs this cleavage; in vivo confirmation is lacking. We demonstrate that primary hepatocytes from tpa KO mice produce less cleaved active PDGF-CC than do wild type hepatocytes, suggesting that tPA is critical for in vitro activation of this growth factor. We developed mice that over-express full-length human PDGF-C in the liver; these mice develop progressive liver fibrosis. To test whether tPA is important for cleavage and activation of PDGF-C in vivo, we intercrossed PDGF-C transgenic (Tg) and tpa knock-out (KO) mice, anticipating that lack of tPA would result in decreased fibrosis due to lack of hPDGF-C cleavage. To measure levels of cleaved, dimerized PDGF-CC in sera, we developed an ELISA that specifically detects cleaved PDGF-CC. We report that the absence of tpa does not affect the phenotype of `PDGF-C Tg mice. PDGF-C Tg mice lacking tPA have high serum levels of cleaved growth factor, significant liver fibrosis, and gene expression alterations similar to those of PDGF-C Tg mice with intact tPA. Furthermore, urokinase plasminogen activator and plasminogen activator inhibitor-1 expression are increased in PDGF-C Tg; tpa KO mice. Our ELISA data suggest a difference between in vitro and in vivo activation of this growth factor, and our mouse model confirms that multiple proteases cleave and activate PDGF-C in vivo.

Rosiglitazone Promotes White Matter Integrity and Long-Term Functional Recovery After Focal Cerebral Ischemia.

BACKGROUND AND PURPOSE: Oligodendrogenesis is essential for white matter repair after stroke. Although agonists of peroxisome proliferator-activated receptors γ confer neuroprotection in models of cerebral ischemia, it is not known whether this effect extends to white matter protection. This study tested the hypothesis that the peroxisome proliferator-activated receptors γ agonist rosiglitazone enhances oligodendrogenesis and improves long-term white matter integrity after ischemia/reperfusion. METHODS: Male adult C57/BL6 mice (25-30 g) were subjected to 60-minute middle cerebral artery occlusion and reperfusion. Rosiglitazone (3 mg/kg) was injected intraperitoneally once daily for 14 days beginning 2 hours after reperfusion. Sensorimotor and cognitive functions were evaluated ≤21 days after middle cerebral artery occlusion. Immunostaining was used to assess infarct volume, myelin loss, and microglial activation. Bromodeoxyuridine (BrdU) was injected for measurements of proliferating NG2(+) oligodendrocyte precursor cells (OPCs) and newly generated adenomatous polyposis coli(+) oligodendrocytes. Mixed glial cultures were used to confirm the effect of rosiglitazone on oligodendrocyte differentiation and microglial polarization. RESULTS: Rosiglitazone significantly reduced brain tissue loss, ameliorated white matter injury, and improved sensorimotor and cognitive functions for at least 21 days after middle cerebral artery occlusion. Rosiglitazone enhanced OPC proliferation and increased the numbers of newly generated mature oligodendrocytes after middle cerebral artery occlusion. Rosiglitazone treatment also reduced the numbers of Iba1(+)/CD16(+) M1 microglia and increased the numbers of Iba1(+)/CD206(+) M2 microglia after stroke. Glial culture experiments confirmed that rosiglitazone promoted oligodendrocyte differentiation, perhaps by promoting microglial M2 polarization. CONCLUSIONS: Rosiglitazone treatment improves long-term white matter integrity after cerebral ischemia, at least, in part, by promoting oligodendrogenesis and facilitating microglial polarization toward the beneficial M2 phenotype.