Circulating progenitor cells are positively associated with cognitive function among overweight/obese children

Recent evidence has indicated that overweight/obese children may experience cognitive and immune dysfunction, but the underlying mechanisms responsible for the association between overweight/obesity, immune dysfunction, and cognition have yet to be established. The present study aimed to identify a novel link between obesity-induced immune system dysregulation and cognition in preadolescent children. A total of 27 male children (age: 8–10 years) were recruited and separated by body mass index (BMI) into healthy weight (HW: 5th–84.9th percentile, n = 16) and overweight/obese (OW: ⩾85th percentile, n = 11) groups. Adiposity was assessed using dual energy X-ray absorptiometry (DXA), and aspects of executive function were assessed using the Woodcock-Johnson III Tests of Cognitive Abilities. Monocyte populations (CD14⁺CD16⁻, CD14⁺CD16⁺) with and without expression of chemokine receptor type 2 (CCR2), and circulating progenitor cells (CPCs: CD34⁺CD45^dim), in peripheral blood were quantified by flow cytometry. CPCs were isolated by flow sorting and cultured for 24 h for collection of conditioned media (CM) that was applied to SH-SY5Y neuroblastomas to examine the paracrine effects of CPCs on neurogenesis. OW had significantly higher quantities of both populations of monocytes (CD14⁺CD16⁻: 57% increase; CD14⁺CD16⁺: 95% increase, both p < 0.01), monocytes expressing CCR2 (CD14⁺CD16⁻CCR2⁺: 66% increase; CD14⁺CD16⁺CCR2⁺: 168% increase, both p < 0.01), and CPCs (47% increase, p < 0.05) than HW. CPCs were positively correlated with abdominal adiposity in OW, and negatively correlated in HW with a significant difference between correlations (p < 0.05). CPC content was positively correlated with executive processes in OW, and negatively correlated in HW with a significant difference in the strength of the correlations between groups (p < 0.05 for correlation between OW and HW). Finally, CPC-CM from OW trended to increase neuroblast viability in vitro relative to HW (1.79 fold, p = 0.07). These novel findings indicate that increased content of CPCs among OW children may play a role in preventing decrements in cognitive function via paracrine mechanisms.     

CCL21-induced calcium transients and proliferation in primary mouse astrocytes: CXCR3-dependent and independent responses

CCL21 is a homeostatic chemokine that is expressed constitutively in secondary lymph nodes and attracts immune cells via chemokine receptor CCR7. In the brain however, CCL21 is inducibly expressed in damaged neurons both in vitro and in vivo and has been shown to activate microglia in vitro, albeit not through CCR7 but through chemokine receptor CXCR3. Therefore, a role for CCL21 in CXCR3-mediated neuron-microglia signaling has been proposed. It is well established that human and mouse astrocytes, like microglia, express CXCR3. However, effects of CCL21 on astrocytes have not been investigated yet. In this study, we have examined the effects of CCL21 on calcium transients and proliferation in primary mouse astrocytes. We show that similar to CXCR3-ligand CXCL10, CCL21 (10^?9 M and 10^?8 M) induced calcium transients in astrocytes, which were mediated through CXCR3. However, in response to high concentrations of CCL21 (10^?7 M) calcium transients persisted in CXCR3-deficient astrocytes, whereas CXCL10 did not have any effect in these cells. Furthermore, prolonged exposure to CXCL10 or CCL21 promoted proliferation of wild type astrocytes. Although CXCL10-induced proliferation was absent in CXCR3-deficient astrocytes, CCL21-induced proliferation of these cells did not significantly differ from wild type conditions. It is therefore suggested that primary mouse astrocytes express an additional (chemokine-) receptor, which is activated at high CCL21 concentrations.     

Lymphocyte infiltration of neocortex and hippocampus after a single brief seizure in mice

Various immune responses have been described in epileptic patients and animal models of epilepsy, but immune responses in brain after a single seizure are poorly understood. We studied immune responses in brain after a single brief generalized tonic–clonic seizure in mice. C57bl/6 mice, either unanesthetized or anesthetized (pentobarbital, ethyl chloride) received either electrical (15–30 mA, 100 Hz, 1 s) or sham stimulation (subcutaneous electrodes over frontal lobe, no current). Electrical stimulation of unanesthetized mice resulted in tonic–clonic convulsions with hind-limb extension (maximal seizure), tonic–clonic convulsions without hind-limb extension (submaximal seizure), or no seizure. In contrast, such stimulation of anesthetized mice did not result in seizure. Mice were killed at 1 h–7 days after seizure. Brains or regions dissected from brain (neocortex, hippocampus, midbrain, cerebellum) of each group were pooled, single cell suspensions prepared, and cells separated according to density. CD4⁺ (CD3⁺CD45^Hi) and CD8⁺ (CD3⁺CD45^Hi) T cell and CD45R⁺ (CD45^Hi) B cell numbers were determined by flow cytometry. At 24 h after a maximal seizure, CD4⁺ and CD8⁺ T cells and CD45R⁺ B cells appeared in brain, reaching peak numbers at 48 h, but were no longer detected at 7 days. CD4⁺ T cells and CD45R⁺ B cells were preferentially found in neocortex compared with hippocampus, whereas CD8⁺ T cells were preferentially found in hippocampus at 24 h after a maximal seizure. In contrast, virtually no lymphocytes were detected in brains of unstimulated or sham stimulated mice, unanesthetized stimulated mice after submaximal or no seizure, and anesthetized stimulated mice at 1 h–7 day. Neither Ly6-G+ neutrophils nor erythrocytes were detected in brains of any animals, nor was there any detectable increase of blood–brain barrier permeability by uptake of Evans Blue dye. The results indicate that lymphocyte entry into brain after a single brief seizure is due to a selective process of recruitment into cortical regions.     

Substantial reduction of naïve and regulatory T cells following traumatic stress

Posttraumatic stress disorder (PTSD) is associated with an enhanced susceptibility to various somatic diseases. However, the exact mechanisms linking traumatic stress to subsequent physical health problems have remained unclear. This study investigated peripheral T lymphocyte differentiation subsets in 19 individuals with war and torture related PTSD compared to 27 non-PTSD controls (n = 14 trauma-exposed controls; n = 13 non-exposed controls). Peripheral T cell subpopulations were classified by their characteristic expression of the lineage markers CD45RA and CCR7 into: naïve (CD45RA⁺ CCR7⁺), central memory (T_CM: CD45RA^? CCR7⁺) and effector memory (T_EM: CD45RA^? CCR7^? and T_EMRA: CD45RA^? CCR7^?) cells. Furthermore, we analyzed regulatory T cells (CD4⁺CD25⁺FoxP3⁺) and ex vivo proliferation responses of peripheral blood mononuclear cells after stimulation with anti-CD3 monoclonal antibody. Results show that the proportion of naïve CD8⁺ T lymphocytes was reduced by 32% (p = 0.01), whereas the proportions of CD3⁺ central (p = 0.02) and effector (p = 0.01) memory T lymphocytes were significantly enhanced (+22% and +34%, respectively) in PTSD patients compared to non-PTSD individuals. To a smaller extent, this effect was also observed in trauma-exposed non-PTSD individuals, indicating a cumulative effect of traumatic stress on T cell distribution. Moreover, PTSD patients displayed a 48% reduction in the proportion of regulatory T cells (p < 0.001). Functionally, these alterations were accompanied by a significantly enhanced (+34%) ex vivo proliferation of anti-CD3 stimulated T cells (p = 0.05). The profoundly altered composition of the peripheral T cell compartment might cause a state of compromised immune responsiveness, which may explain why PTSD patients show an increased susceptibility to infections, and inflammatory and autoimmune diseases.     

Role of resveratrol-induced CD11b(+) Gr-1(+) myeloid derived suppressor cells (MDSCs) in the reduction of CXCR3(+) T cells and amelioration of chronic colitis in IL-10(-/-) mice

Resveratrol, a naturally occurring polyphenol has received significant attention as a potent anti-inflammatory agent. Inflammatory bowel disease (IBD) is a chronic intestinal inflammation caused by hyperactivated effector immune cells that produce proinflammatory cytokines. Myeloid derived suppressor cells (MDSCs) are a heterogeneous population characterized by the co-expression of CD11b⁺ and Gr-1⁺ and have long been known for their immunosuppressive function. We report that resveratrol effectively attenuated overall clinical scores as well as various pathological markers of colitis in IL-10^−/− mice by down regulating Th1 responses. Resveratrol lessened the colitis-associated decrease in body weight and increased levels of serum amyloid A (SAA), CXCL10 and colon TNF-α, IL-6, RANTES, IL-12 and IL-1β concentrations. After resveratrol treatment, the percentage of CXCR3 expressing T cells was decreased in the spleen, mesenteric lymph nodes (MLN), and intestinal lamina propria (LP). However, the percentage and absolute numbers of CD11b⁺ and Gr-1⁺cells in the lamina propria (LP) and spleen were increased after resveratrol treatment as compared with the vehicle treatment. Co-culture of resveratrol-induced CD11b⁺ Gr-1⁺ cells with T cells, attenuated T cell proliferation, and most importantly reduced IFN-γ and GM-CSF production by LP derived T cells from vehicle treated IL-10^−/− mice with chronic colitis. The current study suggests that administration of resveratrol into IL-10^−/− mice induces immunosuppressive CD11b⁺ Gr-1⁺ MDSCs in the colon, which correlates with reversal of established chronic colitis, and down regulation of mucosal and systemic CXCR3⁺ expressing effector T cells as well as inflammatory cytokines in the colon. The induction of immunosuppressive CD11b⁺ Gr-1⁺ cells by resveratrol during colitis is unique, and suggests an as-yet-unidentified mode of anti-inflammatory action of this plant polyphenol.     

Methylmercury chloride exposure aggravates proinflammatory mediators and Notch-1 signaling in CD14+ and CD40+ cells and is associated with imbalance of neuroimmune function in BTBR T+ Itpr3tf/J mice

Autism spectrum disorder (ASD) is a severe neurodevelopmental disorder characterized by deficits in social interaction, communication, and repetitive behaviors. A key role for immune dysfunction has been suggested in ASD. Recent studies have indicated that inflammatory mediators and Notch-1 signaling may contribute to the development of ASD. Methylmercury chloride (MeHgCl) is an environmental pollutant that primarily affects the central nervous system, causing neurological alterations. Its effects on immunological responses have not been fully investigated in ASD. In this study, we examined the influence of MeHgCl exposure on inflammatory mediators and Notch-1 signaling in BTBR T⁺ Itpr3tf/J (BTBR) mice, a model of ASD. We examined the effects of MeHgCl on the IL-6-, GM-CSF-, NF-κB p65-, Notch-1-, and IL-27-producing CD14⁺ and CD40⁺ cells in the spleen. We assessed the effect of MeHgCl on IL-6, GM-CSF, NF-κB p65, Notch-1, and IL-27 mRNA levels in brain tissue. We also measured IL-6, GM-CSF, and NF-κB p65 protein expression levels in brain tissue. MeHgCl exposure of BTBR mice significantly increased IL-6-, GM-CSF-, NF-κB p65-, and Notch-1-, and decreased IL-27-producing CD14⁺, and CD40⁺ cells in the spleen. MeHgCl exposure of BTBR mice upregulated IL-6, GM-CSF, NF-κB p65, and Notch-1, and decreased IL-27 mRNA expression levels in brain tissue. Moreover, MeHgCl resulted in elevated expression of the IL-6, GM-CSF, and NF-κB p65 proteins in brain tissue. Taken together, these results indicate that MeHgCl exposure aggravates proinflammatory mediators and Notch-1 signaling which are associated with imbalance of neuroimmune function in BTBR mice.     

Co-expression of tissue factor and IL-6 in immature endothelial cells of cerebral cavernous malformations

Cerebral cavernous malformations (CCMs) are congenital abnormal clusters of capillaries that are prone to leaking and thought to result from a disorder of endothelial cells. The underlying pathology of CCM is not fully understood. We analyzed the expression of tissue factor (TF) and interleukin-6 (IL-6) in CCMs to determine the association of TF and IL-6 with clinical and pathological findings. Thirteen cases of operative specimens of sporadic CCMs were included in this study. The expression of messenger RNA of TF and IL-6 was assayed and the association with clinical factors was investigated. Then, the distribution of TF and IL-6 was examined with immunofluorescence. The mRNA expression of TF of CCMs was significantly higher than that of the control (p = 0.017), and was correlated with the number of hemorrhage appearances (p = 0.190, ρ = 0.62). The mRNA expression level of IL-6 was significantly correlated with the mRNA expression level of TF (p = 0.034, ρ = 0.58). Examination of immunostained sections indicated that TF⁺ cells were also positive for IL-6, and distributed around normal endothelial cells. Moreover, the TF⁺/IL-6⁺ cells expressed CD31 and VEGFR2. The expressions of IL-6 and TF were correlated, and both were present in the same immature endothelial cells. TF is elevated in CCM and might mediate progressive events. These factors may play a prognostic role in CCM.     

A concerted action of L- and T-type Ca2 + channels regulates locus coeruleus pacemaking

Dysfunction of noradrenergic locus coeruleus (LC) neurons is involved in psychiatric and neurodegenerative diseases and is an early hallmark of Parkinson's disease (PD). The analysis of ion channels underlying the autonomous electrical activity of LC neurons, which is ultimately coupled to cell survival signaling pathways, can lead to a better understanding of the vulnerability of these neurons. In LC neurons somatodendritic Ca^2 + oscillations, mediated by L-type Ca^2 + channels, accompany spontaneous spiking and are linked to mitochondrial oxidant stress. However, the expression and functional implication of low-threshold activated T-type Ca^2 + channels in LC neurons were not yet studied. To this end we performed RT-PCR expression analysis in LC neurons. In addition, we utilized slice patch clamp recordings of in vitro brainstem slices in combination with L-type and T-type Ca^2 + channel blockers. We found the expression of a distinct set of L-type and T-type Ca^2 + channel subtypes mediating a pronounced low-threshold activated Ca^2 + current component. Analyzing spike trains, we revealed that neither L-type Ca^2 + channel nor T-type Ca^2 + channel blockade alone leads to a change in firing properties. In contrast, a combined application of antagonists significantly decreased the afterhyperpolarization amplitude, resulting in an increased firing frequency. Hence, we report the functional expression of T-type Ca^2 + channels in LC neurons and demonstrate their role in increasing the robustness of LC pacemaking by working in concert with Cav1 channels.     

The sympathetic nervous system modulates CD4+Foxp3+ regulatory T cells via noradrenaline-dependent apoptosis in a murine model of lymphoproliferative disease

The sympathetic nervous system (SNS) plays a crucial role in the course and development of autoimmune disease in Fas-deficient lpr/lpr mice. As regulatory T cells (Tregs) are considered important modulators of autoimmune processes, we analyzed the interaction between the SNS and Tregs in this murine model of lymphoproliferative disease. We found that the percentage of Tregs among CD4⁺ T cells is increased in the spleen, lymph nodes, and thymus of lpr/lpr mice as compared to age-matched C57Bl/6J (B6) mice. Furthermore, noradrenaline (NA), the main sympathetic neurotransmitter, induced apoptosis in B6- and lpr/lpr-derived Tregs. NA also reduced the frequency of Foxp3⁺ cells and Foxp3 mRNA expression via β₂-adrenoceptor (β₂-AR)-mediated mechanisms in a concentration and time-dependent manner. Destruction of peripheral sympathetic nerves by 6-hydroxydopamine significantly increased the percentage of Tregs in B6 control mice to an extent comparable to aged-matched lpr/lpr mice. The concentration of splenic NA negatively correlated with the frequency of CD4⁺Foxp3⁺ Tregs. Additionally, 60 days after sympathectomy, a partial recovery of NA concentrations led to Treg percentages comparable to those of intact, vehicle-treated controls. Immunohistochemical analysis of the spleen revealed localization of single Foxp3⁺ Tregs in proximity to NA-producing nerve fibers, providing an interface between Tregs and the SNS. Taken together, our data suggest a relation between the degree of splenic sympathetic innervation and the size of the Treg compartment. While there are few examples of endogenous substances capable of affecting Tregs, our results provide a possible explanation of how the magnitude of the Treg compartment in the spleen can be regulated by the SNS.     

Higher expression of monocyte chemoattractant protein 1 and its receptor in brain tissue of intractable epilepsy patients

We aimed to explore the pathogenesis of monocyte chemoattractant protein-1 (MCP1) and CC chemokine receptor 2 (CCR2) in brain tissue of patients with intractable epilepsy (IE). Hippocampi or temporal lobe tissues were obtained from 40 patients with IE and five patients without IE who had undergone surgical decompression and debridement. The levels of MCP1 and CCR2 were evaluated using immunohistochemistry. Pearson correlation analysis was employed to evaluate the correlation between levels of MCP1 and CCR2 in IE with or without hippocampal sclerosis (HS) and the disease duration, along with age. Higher levels of MCP1 (11.68 ± 4.68% versus 1.72 ± 1.54%) and CCR2 (11.54 ± 4.65% versus 1.52 ± 1.29%; P < 0.05) were observed in IE patients compared to controls. Expression levels of MCP1 (R = 0.867) and CCR2 (R = 0.835) in IE patients with HS were correlated with the disease duration. However, no correlation was found in IE patients without HS. There was also no correlation between levels of MCP1 and CCR2 in IE patients with age, either with HS or without HS. These results suggest that MCP1 and its receptor may play a role in the pathogenesis and progression of IE.     

Freewheel training decreases pro- and increases anti-inflammatory cytokine expression in mouse intestinal lymphocytes

Intestinal inflammation and inflammatory bowel diseases (IBD) may occur due to imbalances in pro- and anti-inflammatory cytokines. Long-term exercise reduces the risk for IBD. The purpose of this study was to determine the effect of long-term wheel running in healthy mice on intestinal lymphocyte (IL) expression of pro- and anti-inflammatory cytokine proteins. In addition, pro- and anti-apoptotic proteins and the percentage of early apoptotic, late apoptotic, and dead IL were measured with wheel running and following acute aerobic exercise. Female C57BL/6 mice were given 16 weeks of wheel running (WR) or a control condition (No WR) and at the end of training were assigned to a single acute treadmill exercise session with sacrifice immediately, 2 h after, or 24 h after completion of exercise, or were not run (sedentary) with respect to the acute treadmill exercise. Intestinal lymphocytes were assessed for pro-(TNF-α, IL-17) and anti-(IL-10) inflammatory, and pleiotropic (IL-6) cytokines, and pro-(caspase 3 and 7, AIF) and anti-(Bcl-2) apoptotic protein expression. The percent of early (Annexin⁺) and late (Annexin⁺PI⁺) apoptotic, and dead (PI⁺) IL was determined. WR mice had lower TNF-α and caspase 7, and higher IL-10 and IL-6 expression in IL than No WR mice. A single exposure to intense aerobic treadmill exercise increased pro-(TNF-α) and anti-(IL-10) inflammatory cytokine and pro-apoptotic protein (caspase 3) expression in IL. The percent of early and late apoptotic, and dead IL were higher after acute exercise. Although long-term voluntary wheel running did not protect against acute exercise-induced changes in IL cytokine expression or apoptosis, there was an overall ‘anti-inflammatory’ effect observed as a result of wheel running in healthy mice.     

Voluntary exercise training in mice increases the expression of antioxidant enzymes and decreases the expression of TNF-α in intestinal lymphocytes

Acute exercise in mice induces intestinal lymphocyte (IL) apoptosis. Freewheel running reduces apoptosis and forced exercise training increases splenocyte antioxidant levels. The purpose of this study was to examine the effect of freewheel running and acute exercise on mouse IL numbers and concentrations of apoptosis and antioxidant proteins and pro-inflammatory cytokines in IL. Female C57BL/6 mice had access to in-cage running wheels (RW) or cages without wheels (NRW) for 16 weeks and were randomized at the end of training to no exercise control (TC) or to treadmill exercise with sacrifice after 90 min of running (TREAD; 30 min, 22 m min^?1; 30 min, 25 m min^?1; 30 min, 28 m min^?1; 2° slope). IL were analyzed for pro-(caspase 3 and 7) and anti-(Bcl-2) apoptotic proteins, endogenous antioxidants (glutathione peroxidase: GPx; catalase: CAT) and the pro-inflammatory cytokine, TNF-α. RW mice had higher cytochrome oxidase (p < 0.001) and citrate synthase (p < 0.01) activities in plantaris and soleus muscles and higher GPx and CAT expression in IL (p < 0.05) (indicative of training) compared with NRW mice. TNF-α expression was lower (p < 0.05) and IL numbers higher (p < 0.05) in RW vs. NRW mice. No training effect was observed for apoptotic protein expression, although TREAD resulted in higher caspase and lower Bcl-2. These results suggest that freewheel running in mice for 16 weeks enhances antioxidant and reduces TNF-α expression in IL but does not reduce pro-apoptotic protein expression after acute exercise. Results are discussed in terms of implications for inflammatory bowel diseases where apoptotic proteins and TNF-α levels are elevated.     

Single low doses of MPTP decrease tyrosine hydroxylase expression in the absence of overt neuron loss

Parkinson's disease (PD) is the second most common age-related neurodegenerative disease. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a prototypical neurotoxicant used in mice to mimic primary features of PD pathology including striatal dopamine depletion and dopamine neuron loss in the substantia nigra pars compacta (SNc). In the literature, there are several experimental paradigms involving multiple doses of MPTP that are used to elicit dopamine neuron loss. However, a recent study reported that a single low dose caused significant loss of dopamine neurons. Here, we determined the effect of a single intraperitoneal injection of one of three doses of MPTP (0.1, 2 and 20 mg/kg) on dopamine neurons, labeled by tyrosine hydroxylase (TH⁺), and total neuron number (Nissl⁺) in the SNc using unbiased stereological counting. Data reveal a significant loss of neurons in the SNc (TH⁺ and Nissl⁺) only in the group treated with 20 mg/kg MPTP. Groups treated with lower dose of MPTP (0.1 and 2 mg/kg) only showed significant loss of TH⁺ neurons rather than TH⁺ and Nissl⁺ neurons. Striatal dopamine levels were decreased in the groups treated with 2 and 20 mg/kg MPTP and striatal terminal markers including, TH and the dopamine transporter (DAT), were only decreased in the groups treated with 20 mg/kg MPTP. These data demonstrate that lower doses of MPTP likely result in loss of TH expression rather than actual dopamine neuron loss in the SN. This finding reinforces the need to measure both total neuron number along with TH⁺ cells in determining dopamine neuron loss.     

Chronic valproate or levetiracetam treatment does not influence cytokine levels in humans

PurposeThere is growing evidence that complex interactions between seizures and the immune system shape the course of epilepsy. However, systematic analyses of the effects of antiepileptic drugs (AED) on the immune system in humans are rare. We performed a prospective study on the influence of the widely used AED valproate and levetiracetam on interictal immunological parameters.Methods36 patients were prospectively included. 15 were started on valproate (5 female (33%), age 54 ± 27 years, 12 (80%) on monotherapy), 21 on levetiracetam (10 female (48%), age 45 ± 19 years, 17 (81%) on monotherapy). Before treatment and after 3 months, we performed a differential blood count and analyzed the distribution of CD3⁺CD4⁺-, CD3⁺CD8⁺- and CD4⁺CD25⁺-leukocyte subsets using flow cytometry. In addition, we determined the concentrations of IL-1β, IL-6, TNF-α and MCP-1 in the peripheral blood using ELISAs.ResultsValproate intake resulted in a significant decrease of the total white blood count (6.96 ± 1.23/nl vs. 6.13 ± 1.57/nl, p = 0.026) and of absolute count and percentage of neutrophils (4.60 ± 1.05/nl vs. 3.69 ± 1.30/nl, p = 0.01; 65.4 ± 7.9% vs. 59.5 ± 11.5%, p = 0.01, respectively). The percentage of CD3⁺CD4⁺-lymphocytes dropped significantly (50.4 ± 10.9% vs. 45.3 ± 12.3%, p = 0.002). Levetiracetam treatment resulted in a decrease of the percentage of CD4⁺CD25⁺-lymphocytes (26.1 ± 8.0% vs. 21.5 ± 9.2%, p = 0.01) but did not significantly alter absolute counts. Neither valproate nor levetiracetam were associated with significant changes in cytokines.ConclusionValproate intake results in profound changes of white blood cell count and subset distribution. Cytokine levels were not influenced by valproate or levetiracetam.     

Lack of association between monocyte protein-1 (MCP-1) –2518 A > G chemoattractant and C–C chemokine receptor 2 (CCR2) Val64Ile polymorphisms and multiple sclerosis in a Tunisian population

Chemokines and their receptors are known to mediate inflammation and tissue damage in autoimmune disorders such as multiple sclerosis (MS). Multiple sclerosis is an inflammatory disease of the central nervous system, characterized by myelin damage and neurological complications. Monocyte chemoattractant protein-1 (MCP-1) interacts with the C–C chemokine receptor 2 (CCR2) and plays a role in the migration of leukocytes into the central nervous system, thus contributing to the T cell-mediated pathogenesis of MS. Genomic DNA obtained from 58 MS patients and 72 healthy controls was tested for the MCP-1 –2518 A > G and CCR2 Val64Ile polymorphisms using polymerase chain reaction–restriction fragment length polymorphism analysis. Neither the MCP-1 –2518G (p = 0.43) nor the CCR2 64Ile (p = 0.52) variant contributed to the risk of MS in Tunisians.     

S100A8 contributes to postoperative cognitive dysfunction in mice undergoing tibial fracture surgery by activating the TLR4/MyD88 pathway

Neuro-inflammation plays a key role in the occurrence and development of postoperative cognitive dysfunction (POCD). Although S100A8 and Toll-like receptor 4 (TLR4) have been increasingly recognized to contribute to neuro-inflammation, little is known about the interaction between S100A8 and TLR4/MyD88 signaling in the process of systemic inflammation that leads to neuro-inflammation. Firstly, we demonstrated that C57BL/6 wide-type mice exhibit cognitive deficit 24 h after the tibial fracture surgery. Subsequently, increased S100A8 and S100A9 expression was found in the peripheral blood mononuclear cells (PBMCs), spleen, and hippocampus of C57BL/6 wide-type mice within 48 h after the surgery. Pre-operative administration of S100A8 antibody significantly inhibited hippocampal microgliosis and improved cognitive function 24 h after the surgery. Secondly, we also observed TLR4/MyD88 activation in the PBMCs, spleen, and hippocampus after the surgery. Compared with those in their corresponding wide-type mice, TLR4^−/− and MyD88^−/− mice showed lower immunoreactive area of microglia in the hippocampal CA3 region after operation. TLR4 deficiency also led to reduction of CD45^hiCD11b⁺ cells in the brain and better performance in both Y maze and open field test after surgery, suggesting a new regulatory mechanism of TLR4-dependent POCD. At last, the co-location of S100A8 and TLR4 expression in spleen after operation suggested a close relationship between them. On the one hand, S100A8 could induce TLR4 activation of CD11b⁺ cells in the blood and hippocampus via intraperitoneal or intracerebroventricular injection. On the other hand, TLR4 deficiency conversely alleviated S100A8 protein-induced hippocampal microgliosis. Furthermore, the increased expression of S100A8 protein in the hippocampus induced by surgery sharply decreased in both TLR4 and MyD88 genetically deficient mice. Taken together, these data suggest that S100A8 exerts pro-inflammatory effect on the occurrence and development of neuro-inflammation and POCD by activating TLR4/MyD88 signaling in the early pathological process of the postoperative stage.     

Early monitoring and quantitative evaluation of macrophage infiltration after experimental traumatic brain injury: A magnetic resonance imaging and flow cytometric analysis

The inflammatory response following traumatic brain injury (TBI) is regulated by phagocytic cells. These cells comprising resident microglia and infiltrating macrophages play a pivotal role in the interface between early detrimental and delayed beneficial effects of inflammation. The aim of the present study was to monitor the early effect of monocyte/phagocytic accumulation and further to explore its kinetics in TBI mice. Localized macrophage population was monitored using ultrasmall superparamagnetic iron oxide (USPIO) nanoparticle enhanced in vivo serial magnetic resonance imaging (MRI). Flow cytometry based gating study was performed to discriminate between resident microglia (Ly6G⁻ CD11b⁺ CD45^low) and infiltrating macrophages (Ly6G⁻ CD11b⁺ CD45^high) at the injury site. The T₂* relaxation analysis revealed that maximum macrophage infiltration occurs between 66 and 72 h post injury (42–48 h post administration of USPIO) at the site of inflammation. This imaging data was well supported by iron oxide specific Prussian blue staining and macrophage specific F4/80 immunohistochemistry (IHC) analysis. Quantitative real-time PCR analysis found significant expression of monocyte chemoattractant protein-1 (MCP-1) at 72 h post injury. Also, we found that flow cytometric analysis demonstrated a 7-fold increase in infiltrating macrophages around 72 h post injuries as compared to control. The MR imaging in combination with flow cytometric analysis enabled the dynamic measurement of macrophage infiltration at the injury site. This study may help in setting an optimal time window to intervene and prevent damage due to inflammation and to increase the therapeutic efficacy.     

Pharmacokinetic,neurochemical, stereological and neuropathological studies on the potential effects of paraquat in the substantia nigra pars compacta and striatum of male C57BL/6J mice

The pharmacokinetics and neurotoxicity of paraquat dichloride (PQ) were assessed following once weekly administration to C57BL/6J male mice by intraperitoneal injection for 1, 2 or 3 weeks at doses of 10, 15 or 25 mg/kg/week. Approximately 0.3% of the administered dose was taken up by the brain and was slowly eliminated, with a half-life of approximately 3 weeks. PQ did not alter the concentration of dopamine (DA), homovanillic acid (HVA) or 3,4-dihydroxyphenylacetic acid (DOPAC), or increase dopamine turnover in the striatum. There was inconsistent stereological evidence of a loss of DA neurons, as identified by chromogenic or fluorescent-tagged antibodies to tyrosine hydroxylase in the substantia nigra pars compacta (SNpc). There was no evidence that PQ induced neuronal degeneration in the SNpc or degenerating neuronal processes in the striatum, as indicated by the absence of uptake of silver stain or reduced immunolabeling of tyrosine-hydroxylase-positive (TH⁺) neurons. There was no evidence of apoptotic cell death, which was evaluated using TUNEL or caspase 3 assays. Microglia (IBA-1 immunoreactivity) and astrocytes (GFAP immunoreactivity) were not activated in PQ-treated mice 4, 8, 16, 24, 48, 96 or 168 h after 1, 2 or 3 doses of PQ.In contrast, mice dosed with the positive control substance, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 10 mg/kg/dose × 4 doses, 2 h apart), displayed significantly reduced DA and DOPAC concentrations and increased DA turnover in the striatum 7 days after dosing. The number of TH⁺ neurons in the SNpc was reduced, and there were increased numbers of degenerating neurons and neuronal processes in the SNpc and striatum. MPTP-mediated cell death was not attributed to apoptosis. MPTP activated microglia and astrocytes within 4 h of the last dose, reaching a peak within 48 h. The microglial response ended by 96 h in the SNpc, but the astrocytic response continued through 168 h in the striatum.These results bring into question previous published stereological studies that report loss of TH⁺ neurons in the SNpc of PQ-treated mice. This study also suggests that even if the reduction in TH⁺ neurons reported by others occurs in PQ-treated mice, this apparent phenotypic change is unaccompanied by neuronal cell death or by modification of dopamine levels in the striatum.