Aquaporin-4 (AQP4) Associations and Array Dynamics Probed by Photobleaching and Single-molecule Analysis of Green Fluorescent Protein-AQP4 Chimeras

The plasma membrane assembly of aquaporin-4 (AQP4) water channels into orthogonal arrays of particles (OAPs) involves interactions of AQP4 N-terminal domains. To study in live cells the site of OAP assembly, the size and dynamics of plasma membrane OAPs, and the heterotetrameric associations of AQP4, we constructed green fluorescent protein (GFP)-labeled AQP4 “long” (M1) and “short” (M23) isoforms in which GFP was inserted at the cytoplasm-facing N or C terminus or between Val-141 and Val-142 in the second extracellular loop of AQP4. The C-terminal and extracellular loop GFP insertions did not interfere with the rapid unrestricted membrane diffusion of GFP-labeled M1 or the restricted diffusion and OAP assembly of GFP-labeled M23. Photobleaching of brefeldin A-treated cells showed comparable and minimally restricted diffusion of M1 and M23, indicating that OAP assembly occurs post-endoplasmic reticulum. Single-molecule step photobleaching and intensity analysis of GFP-labeled M1 in the absence versus presence of excess unlabeled M1 or M23 with an OAP-disrupting mutation indicated heterotetrameric AQP4 association. Time-lapse total internal reflection fluorescence imaging of M23 in live cells at 37 °C indicated that OAPs diffuse slowly (D ∼ 10⁻¹² cm²/s) and rearrange over tens of minutes. Our biophysical measurements in live cells thus reveal extensive AQP4 monomer-monomer and tetramer-tetramer interactions.     

Influence of a brief episode of anesthesia during the induction of experimental brain trauma on secondary brain damage and inflammation

It is unclear whether a single, brief, 15-minute episode of background anesthesia already modulates delayed secondary processes after experimental brain injury. Therefore, this study was designed to characterize three anesthesia protocols for their effect on molecular and histological study endpoints. Mice were randomly separated into groups that received sevoflurane (sevo), isoflurane (iso) or an intraperitoneal anesthetic combination (midazolam, fentanyl and medetomidine; comb) prior to traumatic brain injury (controlled cortical impact, CCI; 8 m/s, 1 mm impact depth, 3 mm diameter). Twenty-four hours after insult, histological brain damage, neurological function (via neurological severity score), cerebral inflammation (via real-time RT-PCR for IL6, COX-2, iNOS) and microglia (via immunohistochemical staining for Iba1) were determined. Fifteen minutes after CCI, the brain contusion volume did not differ between the anesthetic regimens (sevo = 17.9±5.5 mm³; iso = 20.5±3.7 mm³; comb = 19.5±4.6 mm³). Within 24 hours after injury, lesion size increased in all groups (sevo = 45.3±9.0 mm³; iso = 31.5±4.0 mm³; comb = 44.2±6.2 mm³). Sevo and comb anesthesia resulted in a significantly larger contusion compared to iso, which was in line with the significantly better neurological function with iso (sevo = 4.6±1.3 pts.; iso = 3.9±0.8 pts.; comb = 5.1±1.6 pts.). The expression of inflammatory marker genes was not significantly different at 15 minutes and 24 hours after CCI. In contrast, significantly more Iba1-positive cells were present in the pericontusional region after sevo compared to comb anesthesia (sevo = 181±48/mm³; iso = 150±36/mm³; comb = 113±40/mm³). A brief episode of anesthesia, which is sufficient for surgical preparations of mice for procedures such as delivering traumatic brain injury, already has a significant impact on the extent of secondary brain damage.     

Spatial differences in the presence of FOXP3+ and GranzymeB+ T cells between the intra- and extravascular compartments in renal allograft vasculopathy

Background

Allograft vasculopathy (AV) and native atherosclerosis (NA) share the presence of a T-cell mediated inflammatory response, but differ in overall plaque morphology and growth rate. We studied the distribution and frequency of regulatory- and cytotoxic T cells in the arterial intima lesions in both conditions.

Methodology/Principal Findings

The study is based on vessels of 15 explanted human renal allografts with AV and 10 carotid artery plaques obtained at surgery. Distribution and frequency of cytotoxic- and regulatory T cells, as identified by the expression of Granzyme B (GrB) and FOXP3 was established in NA and AV. Furthermore, we compared the distribution of these cells in AV with the perivascular, interstitial renal tissue using immunohistochemistry. The total number of T cells was much higher in AV than in NA lesions (711±135 and 37±8 CD3/mm² respectively, p<0.005, mean, ± SEM). Total numbers of FOXP3⁺ regulatory cells were also significantly increased in AV (36±10 and 0.9±0.3 FOXP3⁺/mm² p<0.05), but relative numbers, expressed as a percentage of the total number of CD3⁺ T cells ((FOXP3⁺/CD3⁺) ×100), were not significantly different (4.6%±0.9 and 2.7%±0.6). GrB⁺ cells were rare in NA, but significantly increased numbers of GrB⁺ cells were found in AV lesions (85±24 and 0.2±0.1 GrB⁺/mm², p<0.05). Perivascular tissues in the allografts showed a higher relative frequency of FOXP3⁺ cells than adjacent intimal lesions (14.0%±2.7 and 4.6%±0.9, respectively, p<0.05), but a lower frequency of GrB⁺ cytotoxic T cells (16.1%±2.7 and 22.6%±3.6, p<0.05).

Conclusions

Similar to NA, AV is characterized by a low frequency of intimal FOXP3⁺ regulatory T cells. Moreover, significant spatial differences exist in the distribution of functional T cell subsets between the intra- and extravascular micro-environments of the graft.     

PLASMA MEMBRANE AND INTERNALIZED IMMUNOGLOBULINS OF LYMPH NODE CELLS STUDIED WITH CONJUGATES OF ANTIBODY OR ITS FAB FRAGMENTS WITH HORSERADISH PEROXIDASE

Normal rat and mouse lymphoid cells were incubated at 0°–4°C for 1 h with purified rabbit or sheep antirat (mouse) immunoglobulin (Ig)-horseradish peroxidase (PO) conjugates or with Fab fragments of antibody coupled with peroxidase. Cells were subsequently washed and incubated in fresh medium, without labeled antibody or Fab fragments for 5–30 min at 20° or 37°C. With the use of the diaminobenzidine (DAB) method, distribution of peroxidase was studied in the light and electron microscopes. Fab fragments of antirat Ig antibody were iodinated with ¹²⁵I and subsequently coupled with horseradish PO. Plasma membrane and internalized immunoglobulins were detected by electron microscope autoradiography and peroxidase cytochemistry. Single- (Fab-PO), and double- ([¹²⁵I]Fab-PO) labeled lymphoid cells showed identical patterns of surface or internal distribution of immunoglobulins. In the electron microscope, Fab-PO conjugates at 0°–4°C resulted in a diffuse specific staining of the plasmalemma of lymphocytes and plasma cells. Most of the small dark lymphocytes (T cells?) did not show plasma membrane Ig. Macrophages did not show plasmalemma staining, but displayed nonspecific cytoplasmic staining after incubation at 20° or 37°C with antibody or Fab-PO conjugates. Lymphocytes and plasma cells, after incubation with antibody-PO conjugates at 0°–4°C, had patchy deposits of oxidized DAB on their plasma membranes. Macrophages, similarly treated, had no plasmalemmal staining. Patch and cap formation on the plasma membrane of lymphocytes and plasma cells was seen regularly after antibody-PO incubation at 37°C. Internalization patterns were different in lymphocytes and plasma cells. In lymphocytes, peroxidase staining was observed in small round or oval vesicles clustered at one pole of the cell (30 min at 37°C). In plasma cells, peroxidase staining was seen in clusters of tubules resembling the Golgi apparatus. Internalization of plasma membrane IgG was less pronounced after antibody-PO labeling as compared to Fab-PO labeling.     

Updates in the pathophysiological mechanisms of Parkinson's disease: Emerging role of bone marrow mesenchymal stem cells

AIM: To explore the approaches exerted by mesenchymal stem cells(MSCs) to improve Parkinson's disease(PD) pathophysiology.METHODS: MSCs were harvested from bone marrowof femoral bones of male rats, grown and propagated in culture. Twenty four ovariectomized animals were classified into 3 groups: Group(1) was control, Groups(2) and(3) were subcutaneously administered with rotenone for 14 d after one month of ovariectomy for induction of PD. Then, Group(2) was left untreated, while Group(3) was treated with single intravenous dose of bone marrow derived MSCs(BM-MSCs). SRY gene was assessed by PCR in brain tissue of the female rats. Serum transforming growth factor beta-1(TGF-β1), monocyte chemoattractant protein-1(MCP-1) and brain derived neurotrophic factor(BDNF) levels were assayed by ELISA. Brain dopamine DA level was assayed fluorometrically, while brain tyrosine hydroxylase(TH) and nestin gene expression were detected by semi-quantitative real time PCR. Brain survivin expression was determined by immunohistochemical procedure. Histopathological investigation of brain tissues was also done.RESULTS: BM-MSCs were able to home at the injured brains and elicited significant decrease in serum TGF-β1(489.7 ± 13.0 vs 691.2 ± 8.0, P 0.05) and MCP-1(89.6 ± 2.0 vs 112.1 ± 1.9, P 0.05) levels associated with significant increase in serum BDNF(3663 ± 17.8 vs 2905 ± 72.9, P 0.05) and brain DA(874 ± 15.0 vs 599 ± 9.8, P 0.05) levels as well as brain TH(1.18 ± 0.004 vs 0.54 ± 0.009, P 0.05) and nestin(1.29 ± 0.005 vs 0.67 ± 0.006, P 0.05) genes expression levels. In addition to, producing insignificant increase in the number of positive cells for survivin(293.2 ± 15.9 vs 271.5 ± 15.9, P 0.05) expression. Finally, the brain sections showed intact histological structure of the striatum as a result of treatment with BM-MSCs. CONCLUSION: The current study sheds light on the therapeutic potential of BM-MSCs against PD pathophysiology via multi-mechanistic actions.     

Treatment with chicken-egg-white or whole-egg extracts maintains and enhances the survival and differentiation of spleen cells

The identification of egg extracts with the ability to maintain and enhance the survival and differentiation of cells would be widely useful in cellular biology research. In this study, we compared the different abilities of spleen cells to survive and differentiate in vivo after permeabilization by five different types of egg extracts. Five types of egg extracts were prepared. The spleen cells from male GFP-transgenic mice were permeabilized by the extracts for 30 min, cultured for 12 days, and then transfused into irradiated female mice. At varying days after transplantation, the percentage of GFP-expressing surviving spleen cells was detected in the peripheral blood by flow cytometry. At 120 days after transplantation, bone marrow cells from the female mice were analyzed for the presence of cells containing the Y chromosome. Surviving GFP-positive spleen cells that had been permeabilized with either chicken-egg-white or whole-egg extracts could be detected in the female mice after transplantation. A lower percentage of GFP-positive cells was also detected after permeabilization by the other extracts tested, and no GFP-positive cells were found in the female mouse transfused with spleen cells permeabilized with Hank’s Buffered Salt Solution (HBSS) as a control. At 120 days after transplantation, the percentage of cells containing a Y chromosome in the bone marrow positively correlated with the percentage of GFP-positive cells in the peripheral blood. After permeabilization by chicken-egg-white or whole-egg extracts, spleen cells demonstrated significantly enhanced survival and differentiation functions compared with the spleen cells treated with the other egg extracts tested. These results show that chicken-egg-white and whole-egg extracts have roles in maintaining and enhancing the survival and differentiation of spleen cells. Therefore, these two types of extracts may be of future use in maintaining the function of stem cells.     

Isolation,expansion and neural differentiation of stem cells from human plucked hair: a further step towards autologous nerve recovery

Stem cells from the adult hair follicle bulge can differentiate into neurons and glia, which is advantageous for the development of an autologous cell-based therapy for neurological diseases. Consequently, bulge stem cells from plucked hair may increase opportunities for personalized neuroregenerative therapy. Hairs were plucked from the scalps of healthy donors, and the bulges were cultured without prior tissue treatment. Shortly after outgrowth from the bulge, cellular protein expression was established immunohistochemically. The doubling time was calculated upon expansion, and the viability of expanded, cryopreserved cells was assessed after shear stress. The neuroglial differentiation potential was assessed from cryopreserved cells. Shortly after outgrowth, the cells were immunopositive for nestin, SLUG, AP-2α and SOX9, and negative for SOX10. Each bulge yielded approximately 1 × 10⁴ cells after three passages. Doubling time was 3.3 (±1.5) days. Cellular viability did not differ significantly from control cells after shear stress. The cells expressed class III β-tubulin (TUBB3) and synapsin-1 after 3 weeks of neuronal differentiation. Glial differentiation yielded KROX20- and MPZ-immunopositive cells after 2 weeks. We demonstrated that human hair follicle bulge-derived stem cells can be cultivated easily, expanded efficiently and kept frozen until needed. After cryopreservation, the cells were viable and displayed both neuronal and glial differentiation potential.     

Tauroursodeoxycholic acid (TUDCA) protects photoreceptors from cell death after experimental retinal detachment

Background

Detachment of photoreceptors from the underlying retinal pigment epithelium is seen in various retinal disorders such as retinal detachment and age-related macular degeneration and leads to loss of photoreceptors and vision. Pharmacologic inhibition of photoreceptor cell death may prevent this outcome. This study tests whether systemic administration of tauroursodeoxycholic acid (TUDCA) can protect photoreceptors from cell death after experimental retinal detachment in rodents.

Methodology/Principal Findings

Retinal detachment was created in rats by subretinal injection of hyaluronic acid. The animals were treated daily with vehicle or TUDCA (500 mg/kg). TUNEL staining was used to evaluate cell death. Photoreceptor loss was evaluated by measuring the relative thickness of the outer nuclear layer (ONL). Macrophage recruitment, oxidative stress, cytokine levels, and caspase levels were also quantified. Three days after detachment, TUDCA decreased the number of TUNEL-positive cells compared to vehicle (651±68/mm² vs. 1314±68/mm², P = 0.001) and prevented the reduction of ONL thickness ratio (0.84±0.03 vs. 0.65±0.03, P = 0.002). Similar results were obtained after 5 days of retinal detachment. Macrophage recruitment and expression levels of TNF-a and MCP-1 after retinal detachment were not affected by TUDCA treatment, whereas increases in activity of caspases 3 and 9 as well as carbonyl-protein adducts were almost completely inhibited by TUDCA treatment.

Conclusions/Significance

Systemic administration of TUDCA preserved photoreceptors after retinal detachment, and was associated with decreased oxidative stress and caspase activity. TUDCA may be used as a novel therapeutic agent for preventing vision loss in diseases that are characterized by photoreceptor detachment.     

Activated platelets in carotid artery thrombosis in mice can be selectively targeted with a radiolabeled single-chain antibody

Background

Activated platelets can be found on the surface of inflamed, rupture-prone and ruptured plaques as well as in intravascular thrombosis. They are key players in thrombosis and atherosclerosis. In this study we describe the construction of a radiolabeled single-chain antibody targeting the LIBS-epitope of activated platelets to selectively depict platelet activation and wall-adherent non-occlusive thrombosis in a mouse model with nuclear imaging using in vitro and ex vivo autoradiography as well as small animal SPECT-CT for in vivo analysis.

Methodology/Principal Findings

LIBS as well as an unspecific control single-chain antibody were labeled with ¹¹¹Indium (¹¹¹In) via bifunctional DTPA ( = ¹¹¹In-LIBS/¹¹¹In-control). Autoradiography after incubation with ¹¹¹In-LIBS on activated platelets in vitro (mean 3866±28 DLU/mm², 4010±630 DLU/mm² and 4520±293 DLU/mm²) produced a significantly higher ligand uptake compared to ¹¹¹In-control (2101±76 DLU/mm², 1181±96 DLU/mm² and 1866±246 DLU/mm²) indicating a specific binding to activated platelets; P<0.05. Applying these findings to an ex vivo mouse model of carotid artery thrombosis revealed a significant increase in ligand uptake after injection of ¹¹¹In-LIBS in the presence of small thrombi compared to the non-injured side, as confirmed by histology (49630±10650 DLU/mm² vs. 17390±7470 DLU/mm²; P<0.05). These findings could also be reproduced in vivo. SPECT-CT analysis of the injured carotid artery with ¹¹¹In-LIBS resulted in a significant increase of the target-to-background ratio compared to ¹¹¹In-control (1.99±0.36 vs. 1.1±0.24; P<0.01).

Conclusions/Significance

Nuclear imaging with ¹¹¹In-LIBS allows the detection of platelet activation in vitro and ex vivo with high sensitivity. Using SPECT-CT, wall-adherent activated platelets in carotid arteries could be depicted in vivo. These results encourage further studies elucidating the role of activated platelets in plaque pathology and atherosclerosis and might be of interest for further developments towards clinical application.     

HMGB‐1 induces c‐kit+ cell microvascular rolling and adhesion via both toll‐like receptor‐2 and toll‐like receptor‐4 of endothelial cells

High-mobility group box 1 (HMGB-1) is a strong chemo-attractive signal for both inflammatory and stem cells. The aim of this study is to evaluate the mechanisms regulating HMGB-1–mediated adhesion and rolling of c-kit⁺ cells and assess whether toll-like receptor-2 (TLR-2) and toll-like receptor-4 (TLR-4) of endothelial cells or c-kit⁺ cells are implicated in the activation of downstream migration signals to peripheral c-kit⁺ cells. Effects of HMGB-1 on the c-kit⁺ cells/endothelial interaction were evaluated by a cremaster muscle model in wild-type (WT), TLR-2 (−/−) and Tlr4 (LPS-del) mice. The mRNA and protein expression levels of endothelial nitric oxide synthase were determined by quantitative real-time PCR and immunofluorescence staining. Induction of crucial adhesion molecules for rolling and adhesion of stem cells and leukocytes were monitored in vivo and in vitro. Following local HMGB-1 administration, a significant increase in cell rolling was detected (32.4 ± 7.1% in ‘WT’ versus 9.9 ± 3.2% in ‘control’, P < 0.05). The number of firmly adherent c-kit⁺ cells was more than 13-fold higher than that of the control group (14.6 ± 5.1 cells/mm² in ‘WT’ versus 1.1 ± 1.0 cells/mm² in ‘control’, P < 0.05). In knockout animals, the fraction of rolling cells did not differ significantly from control levels. Firm endothelial adhesion was significantly reduced in TLR-2 (−/−) and Tlr4 (LPS-del) mice compared to WT mice (1.5 ± 1.4 cells/mm² in ‘TLR-2 (−/−)’ and 2.4 ± 1.4 cells/mm² in ‘Tlr4 (LPS-del)’ versus 14.6 ± 5.1 cells/mm² in ‘WT’, P < 0.05). TLR-2 (−/−) and Tlr4 (LPS-del) stem cells in WT mice did not show significant reduction in rolling and adhesion compared to WT cells. HMGB-1 mediates c-kit⁺ cell recruitment via endothelial TLR-2 and TLR-4.     

In situ dividing and phagocytosing retinal microglia express nestin, vimentin, and NG2 in vivo

Background

Following injury, microglia become activated with subsets expressing nestin as well as other neural markers. Moreover, cerebral microglia can give rise to neurons in vitro. In a previous study, we analysed the proliferation potential and nestin re-expression of retinal macroglial cells such as astrocytes and Müller cells after optic nerve (ON) lesion. However, we were unable to identify the majority of proliferative nestin⁺ cells. Thus, the present study evaluates expression of nestin and other neural markers in quiescent and proliferating microglia in naïve retina and following ON transection in adult rats in vivo.

Methodology/Principal Findings

For analysis of cell proliferation and cells fates, rats received BrdU injections. Microglia in retinal sections or isolated cells were characterized using immunofluorescence labeling with markers for microglia (e.g., Iba1, CD11b), cell proliferation, and neural cells (e.g., nestin, vimentin, NG2, GFAP, Doublecortin etc.). Cellular analyses were performed using confocal laser scanning microscopy. In the naïve adult rat retina, about 60% of resting ramified microglia expressed nestin. After ON transection, numbers of nestin⁺ microglia peaked to a maximum at 7 days, primarily due to in situ cell proliferation of exclusively nestin⁺ microglia. After 8 weeks, microglia numbers re-attained control levels, but 20% were still BrdU⁺ and nestin⁺, although no further local cell proliferation occurred. In addition, nestin⁺ microglia co-expressed vimentin and NG2, but not GFAP or neuronal markers. Fourteen days after injury and following retrograde labeling of retinal ganglion cells (RGCs) with Fluorogold (FG), nestin⁺NG2⁺ microglia were positive for the dye indicating an active involvement of a proliferating cell population in phagocytosing apoptotic retinal neurons.

Conclusions/Significance

The current study provides evidence that in adult rat retina, a specific resident population of microglia expresses proteins of immature neural cells that are involved in injury-induced cell proliferation and phagocytosis while transdifferentiation was not observed.     

Deficiency of vasodilator-stimulated phosphoprotein (VASP) increases blood-brain-barrier damage and edema formation after ischemic stroke in mice

Background

Stroke-induced brain edema formation is a frequent cause of secondary infarct growth and deterioration of neurological function. The molecular mechanisms underlying edema formation after stroke are largely unknown. Vasodilator-stimulated phosphoprotein (VASP) is an important regulator of actin dynamics and stabilizes endothelial barriers through interaction with cell-cell contacts and focal adhesion sites. Hypoxia has been shown to foster vascular leakage by downregulation of VASP in vitro but the significance of VASP for regulating vascular permeability in the hypoxic brain in vivo awaits clarification.

Methodology/Principal Findings

Focal cerebral ischemia was induced in Vasp^−/− mice and wild-type (WT) littermates by transient middle cerebral artery occlusion (tMCAO). Evan''s Blue tracer was applied to visualize the extent of blood-brain-barrier (BBB) damage. Brain edema formation and infarct volumes were calculated from 2,3,5-triphenyltetrazolium chloride (TTC)-stained brain slices. Both mouse groups were carefully controlled for anatomical and physiological parameters relevant for edema formation and stroke outcome. BBB damage (p<0.05) and edema volumes (1.7 mm³±0.5 mm³ versus 0.8 mm³±0.4 mm³; p<0.0001) were significantly enhanced in Vasp^−/− mice compared to controls on day 1 after tMCAO. This was accompanied by a significant increase in infarct size (56.1 mm³±17.3 mm³ versus 39.3 mm³±10.7 mm³, respectively; p<0.01) and a non significant trend (p>0.05) towards worse neurological outcomes.

Conclusion

Our study identifies VASP as critical regulator of BBB maintenance during acute ischemic stroke. Therapeutic modulation of VASP or VASP-dependent signalling pathways could become a novel strategy to combat excessive edema formation in ischemic brain damage.     

A novel animal model of partial optic nerve transection established using an optic nerve quantitative amputator

Background

Research into retinal ganglion cell (RGC) degeneration and neuroprotection after optic nerve injury has received considerable attention and the establishment of simple and effective animal models is of critical importance for future progress.

Methodology/Principal Findings

In the present study, the optic nerves of Wistar rats were semi-transected selectively with a novel optic nerve quantitative amputator. The variation in RGC density was observed with retro-labeled fluorogold at different time points after nerve injury. The densities of surviving RGCs in the experimental eyes at different time points were 1113.69±188.83 RGC/mm² (the survival rate was 63.81% compared with the contralateral eye of the same animal) 1 week post surgery; 748.22±134.75 /mm² (46.16% survival rate) 2 weeks post surgery; 505.03±118.67 /mm² (30.52% survival rate) 4 weeks post surgery; 436.86±76.36 /mm² (24.01% survival rate) 8 weeks post surgery; and 378.20±66.74 /mm² (20.30% survival rate) 12 weeks post surgery. Simultaneously, we also measured the axonal distribution of optic nerve fibers; the latency and amplitude of pattern visual evoke potentials (P-VEP); and the variation in pupil diameter response to pupillary light reflex. All of these observations and profiles were consistent with post injury variation characteristics of the optic nerve. These results indicate that we effectively simulated the pathological process of primary and secondary injury after optic nerve injury.

Conclusions/Significance

The present quantitative transection optic nerve injury model has increased reproducibility, effectiveness and uniformity. This model is an ideal animal model to provide a foundation for researching new treatments for nerve repair after optic nerve and/or central nerve injury.     

Lithium reduces apoptosis and autophagy after neonatal hypoxia�Cischemia

Lithium is used in the treatment of bipolar mood disorder. Reportedly, lithium can be neuroprotective in models of adult brain ischemia. The purpose of this study was to evaluate the effects of lithium in a model of neonatal hypoxic–ischemic brain injury. Nine-day-old male rats were subjected to unilateral hypoxia–ischemia (HI) and 2 mmol/kg lithium chloride was injected i.p. immediately after the insult. Additional lithium injections, 1 mmol/kg, were administered at 24-h intervals. Pups were killed 6, 24 or 72 h after HI. Lithium reduced the infarct volume from 24.7±2.9 to 13.8±3.3 mm³ (44.1%) and total tissue loss (degeneration + lack of growth) from 67.4±4.4 to 38.4±5.9 mm³ (43.1%) compared with vehicle at 72 h after HI. Injury was reduced in the cortex, hippocampus, thalamus and striatum. Lithium reduced the ischemia-induced dephosphorylation of glycogen synthase kinase-3β and extracellular signal-regulated kinase, the activation of calpain and caspase-3, the mitochondrial release of cytochrome c and apoptosis-inducing factor, as well as autophagy. We conclude that lithium could mitigate the brain injury after HI by inhibiting neuronal apoptosis. The lithium doses used were in the same range as those used in bipolar patients, suggesting that lithium might be safely used for the avoidance of neonatal brain injury.     

Multimodality imaging of abnormal vascular perfusion and morphology in preclinical 9L gliosarcoma model

Background

This study demonstrates that a dynamic susceptibility contrast-magnetic resonance imaging (DSC-MRI) perfusion parameter may indicate vascular abnormality in a brain tumor model and reflects an effect of dexamethasone treatment. In addition, X-ray computed tomography (CT) measurements of vascular tortuosity and tissue markers of vascular morphology were performed to investigate the underpinnings of tumor response to dexamethasone.

Methodology/Principal Findings

One cohort of Fisher 344 rats (N = 13), inoculated intracerebrally with 9L gliosarcoma cells, was treated with dexamethasone (i.p. 3 mg/kg/day) for five consecutive days, and another cohort (N = 11) was treated with equal volume of saline. Longitudinal DSC-MRI studies were performed at the first (baseline), third and fifth day of treatments. Relative cerebral blood volume (rCBV) was significantly reduced on the third day of dexamethasone treatment (0.65±.13) as compared to the fifth day during treatment (1.26±.19, p<0.05). In saline treated rats, relative CBV gradually increased during treatment (0.89±.13, 1.00±.21, 1.13±.23) with no significant difference on the third day of treatment (p>0.05). In separate serial studies, microfocal X-ray CT of ex vivo brain specimens (N = 9) and immunohistochemistry for endothelial cell marker anti-CD31 (N = 8) were performed. Vascular morphology of ex vivo rat brains from micro-CT analysis showed hypervascular characteristics in tumors, and both vessel density (41.32±2.34 branches/mm³, p<0.001) and vessel tortuosity (p<0.05) were significantly reduced in tumors of rats treated with dexamethasone compared to saline (74.29±3.51 branches/mm³). The vascular architecture of rat brain tissue was examined with anti-CD31 antibody, and dexamethasone treated tumor regions showed reduced vessel area (16.45±1.36 µm²) as compared to saline treated tumor regions (30.83±4.31 µm², p<0.001) and non-tumor regions (22.80±1.11 µm², p<0.01).

Conclusions/Significance

Increased vascular density and tortuosity are culprit to abnormal perfusion, which is transiently reduced during dexamethasone treatment.     

Cholinesterase activity per unit surface area of conducting membranes

According to theory, the action of acetylcholine (ACh) and ACh-esterase is essential for the permeability changes of excitable membranes during activity. It is, therefore, pertinent to know the activity of ACh-esterase per unit axonal surface area instead of per gram nerve, as it has been measured in the past. Such information has now been obtained with the newly developed microgasometric technique using a magnetic diver. (1) The cholinesterase (Ch-esterase) activity per mm² surface of sensory axons of the walking leg of lobster is 1.2 x 10^-3 µM/hr. (σ = ± 0.3 x 10^-3; SE = 0.17 x 10^-3); the corresponding value for the motor axons isslightly higher: 1.93 x 10^-3 µM/hr. (σ = ± 0.41 x 10^-3; SE = ± 0.14 x 10^-3). Referred to gram nerve, the Ch-esterase activity of the sensory axons is much higher than that of the motor axons: 741 µM/hr. (σ = ± 73.5; SE = ± 32.6) versus 111.6 µM/hr. (σ = ± 28.3; SE = ± 10). (2) The enzyme activity in the small fibers of the stellar nerve of squid is 3.2 x 10^-4 µM/mm²/hr. (σ = ± 0.96 x 10^-4; SE = ± 0.4 x 10^-4). (3) The Ch-esterase activity per mm² surface of squid giant axon is 9.5 x 10^-5 µM/hr. (σ = ± 1.55 x 10^-5; SE = ± 0.38 x 10^-5). The value was obtained with small pieces of carefully cleaned axons after removal of the axoplasm and exposure to sonic disintegration. Without the latter treatment the figurewas 3.85 x 10^-5 µM/mm²/hr. (σ = ± 3.24 x 10^-5; SE = ± 0.93 x 10^-5). The experiments indicate the existence of permeability barriers in the cell wall surrounding part of the enzyme, since the substrate cannot reach all the enzyme even when small fragments of the cell wall are used without disintegration. (4) On the basis of the data obtained, some tentative approximations are made of the ratio of ACh released to Na ions entering the squid giant axon per cm² per impulse.     

Influence of short-term glucocorticoid therapy on regulatory T cells in vivo

Background

Pre- and early clinical studies on patients with autoimmune diseases suggested that induction of regulatory T(T_reg) cells may contribute to the immunosuppressive effects of glucocorticoids(GCs).

Objective

We readdressed the influence of GC therapy on T_reg cells in immunocompetent human subjects and naïve mice.

Methods

Mice were treated with increasing doses of intravenous dexamethasone followed by oral taper, and T_reg cells in spleen and blood were analyzed by FACS. Sixteen patients with sudden hearing loss but without an inflammatory disease received high-dose intravenous prednisolone followed by stepwise dose reduction to low oral prednisolone. Peripheral blood T_reg cells were analyzed prior and after a 14 day GC therapy based on different markers.

Results

Repeated GC administration to mice for three days dose-dependently decreased the absolute numbers of T_reg cells in blood (100 mg dexamethasone/kg body weight: 2.8±1.8×10⁴ cells/ml vs. 33±11×10⁴ in control mice) and spleen (dexamethasone: 2.8±1.9×10⁵/spleen vs. 95±22×10⁵/spleen in control mice), which slowly recovered after 14 days taper in spleen but not in blood. The relative frequency of FOXP3⁺ T_reg cells amongst the CD4⁺ T cells also decreased in a dose dependent manner with the effect being more pronounced in blood than in spleen. The suppressive capacity of T_reg cells was unaltered by GC treatment in vitro. In immunocompetent humans, GCs induced mild T cell lymphocytosis. However, it did not change the relative frequency of circulating T_reg cells in a relevant manner, although there was some variation depending on the definition of the T_reg cells (FOXP3⁺: 4.0±1.5% vs 3.4±1.5%*; AITR⁺: 0.6±0.4 vs 0.5±0.3%, CD127^low: 4.0±1.3 vs 5.0±3.0%* and CTLA4+: 13.8±11.5 vs 15.6±12.5%; * p<0.05).

Conclusion

Short-term GC therapy does not induce the hitherto supposed increase in circulating T_reg cell frequency, neither in immunocompetent humans nor in mice. Thus, it is questionable that the clinical efficacy of GCs is achieved by modulating T_reg cell numbers.     

Salivary extracellular heat shock protein 70 (eHSP70) levels increase after 59 min of intense exercise and correlate with resting salivary secretory immunoglobulin A (SIgA) levels at rest

This study aimed to identify the response of a salivary stress protein, extracellular heat shock protein (eHSP70), to intense exercise and to investigate the relationship between salivary eHSP70 and salivary immunoglobulin A (SIgA) levels in response to exercise. Sixteen healthy sedentary young males (means ± SD 23.8 ± 1.5 years, 172.2 ± 6.4 cm, 68.3 ± 7.4 kg) performed 59 min of cycling exercise at 75 % VO2_max. Saliva and whole blood samples were collected before (Pre), immediately after (Post), and at 1, 2, 3, and 4 h after completion of the exercise (1, 2, 3, and 4 h). The salivary eHSP70 and SIgA levels were measured by enzyme-linked imunosorbent assay (ELISA), and the secretion rates were computed by multiplying the concentration by the saliva flow rate. White blood cells were analyzed using an automated cell counter with a direct-current detection system. The salivary eHSP70 secretion rates were 1.11 ± 0.86, 1.51 ± 1.47, 1.57 ± 1.32, 2.21 ± 2.04, 3.36 ± 2.72, and 6.89 ± 4.02 ng · min⁻¹ at Pre, Post, and 1, 2, 3, and 4 h, respectively. The salivary eHSP70 secretion rate was significantly higher at 4 h than that at Pre, Post, 1, and 3 h (p < 0.05). The SIgA secretion rates were 26.9 ± 12.6, 20.3 ± 10.4, 19.6 ± 11.0, 21.8 ± 12.8, 21.5 ± 11.9, and 21.9 ± 11.7 μg · min⁻¹ at Pre, Post, 1, 2, 3, and 4 h, respectively. The salivary SIgA secretion rate was significantly lower between 1 and 4 h than that at Pre (p < 0.05). There was a positive correlation between salivary eHSP70 and SIgA in both concentration and secretion rates before exercise (p < 0.05). The absolute number of white blood cells significantly increased after exercise, with a maximum at 2 h (p < 0.05). The neutrophil/lymphocyte ratio was significantly increased from 1 to 4 h when compared with that in the Pre samples (p < 0.05). The present study revealed that salivary eHSP70 significantly increased at 4 h after the 59 min of intense exercise in sedentary male subjects. Exercise stress can induce elevated salivary eHSP70 level and upregulate oral immune function partially.     

Toxic effects of resazurin on cell cultures

Resazurin, introduced as a cell viability indicator under the trade name alamarBlue^®, is generally regarded as nontoxic when used according to manufacturer’s suggested shorter-term incubation time specifications. However, problems arise when exposure times are extended to longer-term cultures on the order of days. To assess the effect of resazurin over longer incubation times, MCF7 (HTB-22), MCF10A (CRL-10317), 3T3-L1 (CL-173), and D1 (CRL-12424) cultures were tested with varying amounts of resazurin over 4- and 8-day periods. MCF7, 3T3-L1, and D1 cells cultured for 8 days with 20 % alamarBlue^® had significantly less cell survivability. Specifically, levels of metabolic activity, deoxyribonucleic acid (DNA) concentration, and glucose consumption of the cell lines cultured for 8 days in medium with 20 % alamarBlue^® were significantly lower (p < 0.05) than metabolic activity, DNA concentration, and glucose consumption of MCF7 cells cultured for 8 days in medium with no alamarBlue^®. MCF7, 3T3-L1, and D1 cells used less glucose at concentrations as low as 5 %. Data also suggests the toxic effects are more pronounced in the cancerous cell line as compared to the noncancerous cells.