Number Of CD11b-positive Cells Research Articles

Abstract WP215: Increase In CD11b Positive Inflammatory Cells In Cardioembolic And Cryptogenic Strokes: A Study Of Thrombi From Stroke Patients

Introduction: Around 25-40% of ischemic strokes are cryptogenic or strokes of undetermined etiology. Determination of stroke etiology can be challenging, requiring extensive diagnostic testing including long term monitoring for occult atrial fibrillation. Recent studies suggest that inflammation (monocytes/ macrophages driven) and fibrosis in the atrial substrate leads to atrial fibrillation/ cardioembolic strokes. Therefore, we hypothesized that clots collected from patients with cardioembolic stroke during thrombectomy will have increased myeloid derived inflammatory cells as compared to other stroke etiologies. Methods: 54 thrombi were obtained during mechanical thrombectomy in patients with ischemic strokes after consent. They were formalin fixed, embedded, sliced and stained with CD11b, a marker for myeloid derived cells and some lymphoid cells. Mean of five 20x images from two slices per patient was quantified by image J and used for analysis. The relationship between stroke etiology (TOAST) and number of CD11b positive cells was assessed using Mann-Whitney U test and multilinear regression models. Results: Mean patient age was 64.2±12.5 years, 33.3% were women, 81.5% had hypertension, 22.2% had diabetes mellitus, and 37% had hyperlipidemia. Thrombi with cardioembolic etiology had significantly higher number of CD11b positive cells (248.2±137.4, n=27) as compared to strokes from large artery atherosclerosis (133.3±95.7, n=11), p=0.02. Cryptogenic strokes also had significantly higher number of CD11b positive cells, (212.1± 104.8, n=13) when compared to strokes from large artery atherosclerosis, p=0.04, n=11, even after controlling for hypertension, hyperlipidemia, heart disease, diabetes mellitus and age. No difference was seen in CD11b positive cell numbers between cardioembolic and cryptogenic strokes, p=0.4. Conclusion: Cardioembolic and cryptogenic stroke thrombi have increased CD11b positive cells as compared to strokes from large artery atherosclerosis. The shared histology of cardioembolic and cryptogenic strokes suggests that many of the cryptogenic strokes may be due to undiagnosed cardioembolism. Future studies using flow cytometry on thrombi can be a novel area of research for stroke etiology.

Myonuclear apoptosis via cleaved caspase-3 upregulation is related to macrophage accumulation underlying immobilization-induced muscle fibrosis.

Although macrophage accumulation plays a key role in the development of immobilization-induced muscle fibrosis, the underlying mechanisms remain unclear. Therefore, we focused on the alterations of myonuclear apoptosis via cleaved caspase-3, and investigated whether these changes may be related to macrophage accumulation. Eight-week-old Wistar rats were divided into immobilization and control groups, and the soleus muscles were selected for analysis. The mRNA and protein expression of collagen and the number of CD11b-positive cells were significantly higher in the immobilized rats than in the control rats at 1 and 2 weeks. TdT-mediated dUTP nick end-labeling (TUNEL)-positive myonuclei counts in 1- and 2-week control rats were 0.2 ± 0.1 and 0.2 ± 0.5, whereas they were 1.0 ± 0.6 and 1.1 ± 0.5 in 1- and 2-week immobilized rats. The cleaved caspase-3 protein expressions in 1- and 2-week control rats were 0.2 ± 0.1 and 0.2 ± 0.1, whereas they were 0.5 ± 0.1 and 0.4 ± 0.2 in 1- and 2-week immobilized rats. TUNEL-positive myonuclei counts and cleaved caspase-3 protein expression were significantly higher in immobilized rats than in control rats at 1 and 2 weeks. The numbers of myonuclei in 1- and 2-week control rats were 2.8 ± 0.1 and 2.6 ± 0.4, whereas they were 2.2 ± 0.4 and 2.2 ± 0.2 in 1- and 2-week immobilized rats. The numbers of myonuclei were significantly lower in immobilized than in control rats at both time-points. Myonuclear apoptosis via the upregulation of cleaved caspase-3 might induce macrophage accumulation. These alterations are related to immobilization-induced muscle fibrosis.

Mechanisms of macrophages accumulation in the process of immobilized-induced muscle contracture in rats

Skeletal muscle fibrosis is the main pathology of immobilized-induced muscle contracture. Our previous studies revealed that upregulation of interleukin-1β/transforming growth factor-β1 associated with macrophages accumulation was related to the development of skeletal muscle fibrosis. However, the mechanisms underlying the increase in the number of macrophages in the immobilized soleus muscle are unclear. In this study, we investigated the mechanisms of macrophages accumulation in the immobilized soleus muscle of rat ankle- joint contracture models. Rats were divided into immobilization and control groups. In the immobilization group, both ankle joints of each rats were immobilized in full plantar flexion with plaster casts for 1 and 2 weeks. To assess alterations of macrophages, the number of CD11b-positive cells and the monocyte chemotactic protein (MCP)-1 mRNA expression were measured. Additionally, to assess alterations of the myocyte, the number of myonuclei and TUNEL-positive cells, cross-sectional area (CSA) of myofibers, and the myonuclear domain size were measured. Immobilization resulted in a significant increase in the number of CD11b-positive cells and the MCP-1 mRNA expression at 1 and 2 weeks. The number of myonuclei and the CSA of myofibers were significantly decreased in the immobilization group compared to the control group at 1 and 2 weeks. Only the CSA of myofibers significantly decreased, depending on the immobilization period. The myonuclear domain size was significantly lower in the immobilization group at 2 weeks compared to the control group. TUNEL-positive cells were significantly increased in the immobilization group compared to the control group at 1 and 2 weeks. These results indicated that joint immobilization after 1 week induced macrophages accumulation and reduced the number of myonuclei associated with myocyte apoptosis, which caused a decrease in the CSA of myofibers. These changes suggest that immobilized-induced muscle fiber atrophy following myocyte apoptosis induce macrophage accumulation.

Role of Toll-like receptor 4 in mechanical ventilation-induced activation of microglias in hippocampi of mice

Objective To evaluate the role of Toll-like receptor 4(TLR4)in mechanical ventilation-induced activation of microglias in the hippocampi of mice. Methods Forty-two healthy male TLR4 gene knocked out C57BL/10ScNJNju mice and 42 wild type C57BL/6 mice, aged 10-12 weeks, weighing 20-25 g, were used in the study.The mice of either group were divided into 2 subgroups(n=21 each)using a random number table: spontaneous breathing subgroup(subgroup SB)and mechanical ventilation subgroup(subgroup MV). In subgroup SB, the mice were exposed to isoflurane for 6 h in an anesthesia chamber.After tracheal intubation, the mice were mechanically ventilated for 6 h when anesthesia was maintained with isoflurane in subgroup MV.Twelve mice were selected from each subgroup at 1 and 3 days after ventilation, fear conditioning test was performed to assess the cognitive function, and the rate of freezing time was recorded.Three mice in each subgroup were sacrificed at 1 day after ventilation, and the brains were removed to observe the morphological changes of microglias(by double immunofluorescent staining)and to count CD11b positive cells and co-expression of TLR4 with CD11b(TLR4/CD11b)positive cells in hippocampal microglias.Six mice in each subgroup were sacrificed at 1 day after ventilation, and the hippocampi were isolated for determination of the content of tumor necrosis factor-alpha(TNF-α)in hippocampal tissues by enzyme-linked immunosorbent assay. Results Compared with subgroup SB of wild type group, the rate of freezing time was significantly decreased at 1 and 3 days after ventilation, and the number of CD11b positive cells and TLR4/CD11b positive cells were increased, and the content of TNF-α was increased in subgroup MV of wild type group(P<0.05), and the number of CD11b positive cells and content of TNF-α were significantly increased in subgroup MV of gene knockout group(P<0.05). Compared with subgroup SB of gene knockout group, the rate of freezing time was significantly decreased at 1 and 3 days after ventilation, and the number of CD11b positive cells and content of TNF-α were increased in subgroup MV of gene knockout group(P<0.05). Compared with subgroup MV of wild type group, the rate of freezing time was significantly increased at 1 and 3 days after ventilation, and the number of CD11b positive cells and content of TNF-α were decreased in subgroup MV of gene knockout group(P<0.05). Conclusion The mechanism by which mechanical ventilation induces activation of microglias in hippocampi is partially related to TLR4 in mice. Key words: Toll like receptor 4; Respiration, artificial; Hippocampus; Microglia

Age-Related Decrease in Glucagon-Like Peptide-1 in Mouse Prefrontal Cortex but Not in Hippocampus Despite the Preservation of Its Receptor

Glucagon-like peptide-1 receptor (GLP-1R) agonist treatment has the potential to be a novel therapeutic treatment for Alzheimer’s disease (AD). We previously reported that exendin-4, a Gαs protein-coupled GLP-1R agonist, up-regulates the membrane AMPA receptor GluR1 subunit in the neocortex. However, it is uncertain whether GLP-1R agonists have an advantage as an AD treatment target compared with other Gαs protein-coupled receptors. Here we show that both the protein level of proglucagon, a precursor of GLP-1, and the immunoreactivity level of GLP-1 are significantly decreased in the medial prefrontal cortex (mPFC) of aged mice (14 months old) compared with young (3 weeks old) or adult (6 months old) mice, but not in area CA1, the dentate gyrus (DG) nor in the nucleus of the solitary tract. However, the protein and immunoreactivity levels of GLP-1R in the mPFC, DG and hippocampal CA1 and CA3 areas were preserved in the aged mice. We then confirmed whether the age-dependent decrease in GLP-1 in the mPFC was associated with the activity level or the number of microglial cells in the mPFC. Co-staining of CD11b and GLP-1 in the mPFC revealed that the number of CD11b-positive cells was increased in the aged mice. Moreover, lipopolysaccharide (LPS) injection increased the number of CD11b-positive cells in the mPFC, but the number of GLP-1-positive cells was unchanged. However, the number of CD11b-positive cells that co-localized with GLP-1R in the mPFC is increased by LPS and aging. Because the GLP-1R is preserved in aged mPFC, but the amount of GLP-1 produced in the brain region is diminished, and spatial cognitive memory was impaired in aged mice, we propose that treatment with GLP-1 analogues has great promise for rescuing and ameliorating the age-related mPFC-dependent decline of cognitive functions.

LASIK following Small Incision Lenticule Extraction (SMILE) Lenticule Re-Implantation: A Feasibility Study of a Novel Method for Treatment of Presbyopia

Presbyopia remains a major visual impairment for patients, who have previously undergone laser refractive correction and enjoyed unaided distance vision prior to the onset of presbyopia. Corneal stromal volume restoration through small incision lenticule extraction (SMILE) lenticule re-implantation presents an opportunity for restoring the patients’ non-dominant eye to previous low myopia to achieve a monovision. In this study, we investigated the feasibility of performing LASIK after lenticule re-implantation as a method to create presbyopic monovision. A -6.00D SMILE correction was performed in 9 rabbit eyes. The lenticules were cryopreserved for 14 days and re-implanted. Five weeks later, 3 of these eyes underwent LASIK for -5.00D correction (RL group); 3 underwent LASIK flap creation, which was not lifted (RN); and no further procedures were performed on the remaining 3 eyes. These groups were compared with 3 eyes that underwent standard LASIK for a -5.00D correction (LO); 3 that underwent creation of non-lifted flap (LN); and 3 non-operated eyes. Rabbits were euthanized 1 day post-surgery. Tissue responses were analyzed by immunohistochemistry, slit lamp and in vivo confocal microscopy (IVCM). Intrastromal irregularities and elevated reflectivity levels of the excimer-ablated plane were observed on slit lamp and IVCM, respectively in the RL group. The results were comparable (P = 0.310) to IVCM findings in the LO group. RL and LO groups showed similar fibronectin expression levels, number of CD11b-positive cells (P = 0.304) and apoptotic cells (P = 0.198). There was no difference between the RN and LN groups in reflectivity levels (P = 0.627), fibronectin expression levels, CD11b-positive cells (P = 0.135) and apoptotic cells (P = 0.128). LASIK can be performed following lenticule re-implantation to create presbyopic monovision. The tissue responses elicited after performing LASIK on corneas that have undergone SMILE and subsequent lenticule re-implantation are similar to primary procedure.

Pharmacologic resuscitation for hemorrhagic shock combined with traumatic brain injury

We have previously demonstrated that valproic acid (VPA), a histone deacetylase inhibitor, can improve survival after hemorrhagic shock (HS), protect neurons from hypoxia-induced apoptosis, and attenuate the inflammatory response. We have also shown that administration of 6% hetastarch (Hextend [Hex]) after traumatic brain injury (TBI) decreases brain swelling, without affecting size of the lesion. This study was performed to determine whether addition of VPA to Hex would decrease the lesion size in a clinically relevant large animal model of TBI + HS. Yorkshire swine (42-50 kg) were instrumented to measure hemodynamic parameters, intracranial pressure, and brain tissue oxygenation. A custom-designed, computer-controlled cortical impact device was used to create a TBI through a 20-mm craniotomy: 15-mm cylindrical tip impactor at 4-m/s velocity, 100-millisecond dwell time, and 12-mm penetration depth. Volume-controlled hemorrhage was started (40% blood volume) concurrent with the TBI. After 2 hours of shock, animals were randomized to one of three resuscitation groups (n = 7 per group) as follows: (1) isotonic sodium chloride solution; (2) 6% hetastarch, Hex; and (3) Hex and VPA 300 mg/kg (Hex + VPA). Volumes of Hex matched the shed blood, whereas that of the isotonic sodium chloride solution was three times the volume. VPA treatment was started after an hour of shock. After 6 hours of postresuscitation monitoring, brains were sectioned into 5-mm slices and stained with 2, 3, 5-Triphenyltetrazolium chloride to quantify the lesion size (mm) and brain swelling (percent change compared with uninjured side). Levels of acetylated histone H3 were determined to quantify acetylation, and myeloperoxidase and interleukine-1β (IL-1β) levels were measured as markers of brain inflammation. Combination of 40% blood loss with cortical impact and a period of shock (2 hours) and resuscitation resulted in a highly reproducible brain injury. Lesion size and brain swelling in the Hex + VPA group (1,989 [156.8] mm, and 19% [1.6%], respectively) were significantly smaller than the isotonic sodium chloride solution group (3,335 [287.9] mm and 36% [2.2%], respectively). Hex alone treatment significantly decreased the swelling (27% [1.6%]) without reducing the lesion size. The number of CD11b-positive cells as well as myeloperoxidase and IL-1 levels in the brains were significantly reduced by the VPA treatment. In a combined HS and TBI model, treatment with artificial colloid (Hex) improves hemodynamic parameters and reduces swelling, without affecting the actual size of the brain lesion. Addition of VPA effectively reduces both the size of brain lesion and associated swelling by attenuating the inflammatory response.

Toll-Like Receptor 4 Is a Regulator of Monocyte and Electroencephalographic Responses to Sleep Loss

Sleep loss triggers changes in inflammatory signaling pathways in the brain and periphery. The mechanisms that underlie these changes are ill-defined. The Toll-like receptor 4 (TLR4) activates inflammatory signaling cascades in response to endogenous and pathogen-associated ligands known to be elevated in association with sleep loss. TLR4 is therefore a possible mediator of some of the inflammation-related effects of sleep loss. Here we describe the baseline electroencephalographic sleep phenotype and the biochemical and electroencephalographic responses to sleep loss in TLR4-deficient mice. TLR4-deficient mice and wild type controls were subjected to electroencephalographic and electromyographic recordings during spontaneous sleep/wake cycles and during and after sleep restriction sessions of 3, 6, and 24-h duration, during which sleep was disrupted by an automated sleep restriction system. Relative to wild type control mice, TLR4-deficient mice exhibited an increase in the duration of the primary daily waking bout occurring at dark onset in a light/dark cycle. The amount of time spent in non-rapid eye movement sleep by TLR4-deficient mice was reduced in proportion to increased wakefulness in the hours immediately after dark onset. Subsequent to sleep restriction, EEG measures of increased sleep drive were attenuated in TLR4-deficient mice relative to wild-type mice. TLR4 was enriched 10-fold in brain cells positive for the cell surface marker CD11b (cells of the monocyte lineage) relative to CD11b-negative cells in wild type mouse brains. To assess whether this population was affected selectively by TLR4 knockout, flow cytometry was used to count F4/80- and CD45-positive cells in the brains of sleep deprived and time of day control mice. While wild-type mice exhibited a significant reduction in the number of CD11b-positive cells in the brain after 24-h sleep restriction, TLR4-deficient mice did not. These data demonstrate that innate immune signaling pathways active in the monocyte lineage, including presumably microglia, detect and mediate in part the cerebral reaction to sleep loss.

Early Corneal Wound Healing and Inflammatory Responses after Refractive Lenticule Extraction (ReLEx)

To compare the early corneal wound repair and inflammatory responses after refractive lenticule extraction (ReLEx) and LASIK. Eighteen rabbits underwent ReLEx and another 18 underwent LASIK. Each group was divided into three subgroups of six rabbits each and these were subjected to refractive corrections of -3.00 diopters (D), -6.00 D, and -9.00 D. Slit lamp photography, anterior segment optical coherence tomography (AS-OCT), corneal topography, and in vivo confocal microscopy were performed 1 day after surgery. After euthanatization, the corneas were subjected to immunofluorescent staining for fibronectin, CD11b, Ki-67, and TUNEL assay. On slit lamp microscopy, all corneas appeared clear pre- and postoperatively in both ReLEx and LASIK eyes. Corneal topography showed a more significant corneal flattening after LASIK than after ReLEx as the degree of correction was increased (P = 0.916 after -3.00 D correction to P = 0.097 after -9.00 D correction). In vivo confocal microscopy showed less light-scattering particles at the flap interface after ReLEx compared with LASIK. Immunostaining of fibronectin showed a less abundant expression in corneas that underwent ReLEx than LASIK. The differences became more marked as the power of correction was increased. Similar trend was seen in the number of CD11b-positive cells (P = 0.476 after -3.00 D correction to P < 0.001 after -9.00D correction). There was no marked disparity observed in cell death and proliferation between post-ReLEx and -LASIK eyes. This study has shown that the ReLEx procedure may result in less topographic changes, inflammation, and early extracellular matrix deposition than LASIK, especially at high refractive correction.

The investigation of macrophage infiltration in the early phase of ischemic acute renal failure in mice

Background/AimsInflammation plays a key role in ischemic acute renal failure (ARF). The present study investigated the infiltration of macrophages in the early phase of ischemic ARF in mice.MethodsIschemic ARF was induced by renal clamping for 22 min, while the control mice underwent sham surgery (no clamping). The serum creatinine and blood urea nitrogen (BUN) levels were measured in the control and post-ischemia mice. Immunofluorescence staining was used to measure the number of CD 11b-positive cells in the kidney tissue sections to determine the amount of post-ischemic macrophage infiltration. Lipo-Cl2MBP (clodronate) for macrophages depletion was injected via a tail vein 5 d before ischemia induction and again 2 d before ischemia induction.ResultsThe study found that the post-ischemia mice had higher levels of serum creatinine and BUN at 16 and 24 h compared to the controls. Immunofluorescence staining showed there were more macrophages in the post-ischemic tissue at 2, 8, 16 and 24 h compared to the control tissue, and that most of these macrophages were located in the outer medulla. The mice treated with clodronate prior to ischemia induction were found to have lower levels of serum creatinine compared to those mice that weren't treated with clodronate.ConclusionsThere was significant infiltration of macrophages from the early phase of ischemic ARF, and this peaked at 16-24 h. Macrophage depletion using clodronate was protective against ischemic ARF.

Modulation of ionizing radiation-induced apoptosis and cell cycle arrest by all-trans retinoic acid in promyelocytic leukemia cells (HL-60).

Acute promyelocytic leukemia is characterized by a block of myeloid differentiation. The incubation of cells with 1 μmol/l all-trans retinoic acid (ATRA) for 72 h induced differentiation of HL-60 cells and increased the number of CD11b positive cells. Morphological and functional changes were accompanied by a loss of proliferative capacity. Differentiation caused by preincubation of leukemic cells HL-60 with ATRA is accompanied by loss of clonogenicity (control cells: 870 colonies/103 cells, cells preincubated with ATRA: 150 colonies/103 cells). D0 for undifferentiated cells was 2.35 Gy, for ATRA differentiated cells 2.46 Gy. Statistical comparison of clonogenity curves indicated that in the whole range 0.5-10 Gy the curves are not significantly different, however, in the range 0.5-3 Gy ATRA differentiated cells were significantly more radioresistant than non-differentiated cells. When HL-60 cells preincubated with 1 μmol/l ATRA were irradiated by a sublethal dose of 6 Gy, more marked increase of apoptotic cells number was observed 24 h after irradiation and the surviving cells were mainly in the G1 phase of the cell cycle, while only irradiated cells were accumulated in G2 phase. Our results imply that preincubation of cells with ATRA accelerates apoptosis occurrence (24 h) after irradiation by high sublethal dose of 6 Gy. Forty-eight hours after 6 Gy irradiation, late apoptotic cells were found in the group of ATRA pretreated cells, as determined by APO2.7 positivity. This test showed an increased effect (considering cell death induction) in comparison to ATRA or irradiation itself.