Parallel Time Course Research Articles

Microcirculation-driven mitochondrion dysfunction during the progression of experimental sepsis

Sepsis is accompanied by a less-known mismatch between hemodynamics and mitochondrial respiration. We aimed to characterize the relationship and time dependency of microcirculatory and mitochondrial functions in a rodent model of intraabdominal sepsis. Fecal peritonitis was induced in rats, and multi-organ failure (MOF) was evaluated 12, 16, 20, 24 or 28 h later (n = 8/group, each) using rat-specific organ failure assessment (ROFA) scores. Ileal microcirculation (proportion of perfused microvessels (PPV), microvascular flow index (MFI) and heterogeneity index (HI)) was monitored by intravital video microscopy, and mitochondrial respiration (OxPhos) and outer membrane (mtOM) damage were measured with high-resolution respirometry. MOF progression was evidenced by increased ROFA scores; microcirculatory parameters followed a parallel time course from the 16th to 28th h. Mitochondrial dysfunction commenced with a 4-h time lag with signs of mtOM damage, which correlated significantly with PPV, while no correlation was found between HI and OxPhos. High diagnostic value was demonstrated for PPV, mtOM damage and lactate levels for predicting MOF. Our findings indicate insufficient splanchnic microcirculation to be a possible predictor for MOF that develops before the start of mitochondrial dysfunction. The adequate subcellular compensatory capacity suggests the presence of mitochondrial subpopulations with differing sensitivity to septic insults.

The transported cations impose differences in the thermostability of the gastric H,K-ATPase. A kinetic analysis

This work analyses the thermostability of a membrane protein, the gastric H,K-ATPase, by means of a detailed kinetic characterization of its inactivation process, which showed to exhibit first-order kinetics. We observed parallel time courses for the decrease of ATPase activity, the decrease of the autophosphorylation capacity and the loss of tertiary structure at 49 °C. Higher temperatures were required to induce a significant change in secondary structure. The correspondence between the kinetics of Trp fluorescence measured at 49 °C and the decrease of the residual activity after heating at that temperature, proves the irreversibility of the inactivation process. Inactivation proceeds at different rates in E1 or E2 conformations. The K+-induced E2 state exhibits a lower inactivation rate; the specific effect is exerted with a K0.5 similar to that found at 25 °C, providing a further inkling that K+ occlusion by the H,K-ATPase is not really favoured. Increasing [H+] from pH 8 to pH 7, which possibly shifts the protein to E1, produces a subtle destabilizing effect on the H,K-ATPase. We performed a prediction of potential intramolecular interactions and found that the differential stability between E1 and E2 may be mainly explained by the higher number of hydrophobic interactions in the α- and β-subunits of E2 conformation.

Thalamic and neocortical differences in the relationship between the time course of delta and sigma power during NREM sleep in humans.

Sleep spindles and slow waves are the hallmarks of non-rapid eye movement (NREM) sleep and are produced by the dynamic interplay between thalamic and cortical regions. Several studies in both human and animal models have focused their attention on the relationship between electroencephalographic (EEG) spindles and slow waves during NREM, using the power in the sigma and delta bands as a surrogate for the production of spindles and slow waves. A typical report is an overall inverse relationship between the time course of sigma and delta power as measured by a single correlation coefficient both within and across NREM episodes. Here we analysed stereotactically implanted intracerebral electrode (Stereo-EEG [SEEG]) recordings during NREM simultaneously acquired from thalamic and from several neocortical sites in six neurosurgical patients. We investigated the relationship between the time course of delta and sigma power and found that, although at the cortical level it shows the expected inverse relationship, these two frequency bands follow a parallel time course at the thalamic level. Both these observations were consistent across patients and across different cortical as well as thalamic regions. These different temporal dynamics at the neocortical and thalamic level are discussed, considering classical as well as more recent interpretations of the neurophysiological determinants of sleep spindles and slow waves. These findings may also help understanding the regulatory mechanisms of these fundamental sleep EEG graphoelements across different brain compartments.

Disappearance of cerebrovascular laminin immunoreactivity as related to the maturation of astroglia in rat brain

The present paper provides novel findings on the temporo-spatial correlation of perivascular laminin immunoreactivity with the early postnatal astrocyte development. The cerebrovascular laminin immunoreactivity gradually disappears during development. The fusion of the glial and vascular basal laminae during development makes the laminin epitopes inaccessible for antibody molecules (Krum et al., 1991, Exp Neurol 111:151). The fusion is supposed to correlate with the maturation of the glio-vascular connections. Glial development was followed by immunostaining for GFAP (glial fibrillary acidic protein), S100 protein, glutamine synthetase as glial markers and for nestin to visualize the immature glial structures. Our investigation focused on the period from postnatal day (P)2 to P16, on the dorso-parietal pallium. In the wall of the telencephalon the laminin immunoreactivity disappeared between P5 and P10; in subcortical structures it persisted to P12 or even to P16. Its disappearance overlapped the period when GFAP-immunopositive astrocytes were taking the place of radial glia. Despite the parallel time courses, however, the spatial patterns of the two processes were just the opposite: disappearance of the laminin immunoreactivity progressed from the middle zone whereas the appearance of GFAP from the pial surface and the corpus callosum. Rather, the regression of the vascular laminin immunoreactivity followed the progression of the immunoreactivities of glutamine synthetase and S100 protein. Therefore, the regression really correlates with a ‘maturation’ of astrocytes which, however, affects other astrocyte functions rather than cytoskeleton.

Pressure balance and imbalance in the optic nerve chamber: The Beijing Intracranial and Intraocular Pressure (iCOP) Study.

To determine the interdependence of intracranial pressure (ICP) and intraocular pressure (IOP) and how it affects optic nerve pressures, eight normal dogs were examined using pressure-sensing probes implanted into the left ventricle, lumbar cistern, optic nerve subarachnoid space in the left eye, and anterior chamber in the left eye. This allowed ICP, lumbar cistern pressure (LCP), optic nerve subarachnoid space pressure (ONSP) and IOP to be simultaneously recorded. After establishing baseline pressure levels, pressure changes that resulted from lowering ICP (via shunting cerebrospinal fluid (CSF) from the ventricle) were recorded. At baseline, all examined pressures were different (ICP<LCP<ONSP), but correlated (P>0.001). As ICP was lowered during CSF shunting, IOP also dropped in a parallel time course so that the trans-lamina cribrosa gradient (TLPG) remained stable (ICP-IOP dependent zone). However, once ICP fell below a critical breakpoint, ICP and IOP became uncoupled and TLPG changed as ICP declined (ICP-IOP independent zone). The optic nerve pressure gradient (ONPG) and trans-optic nerve pressure gradient (TOPG) increased linearly as ICP decreased through both the ICP-IOP dependent and independent zones. We conclude that ICP and IOP are coupled in a specific pressure range, but when ICP drops below a critical point, IOP and ICP become uncoupled and TLPG increases. When ICP drops, a rise in the ONPG and TOPG creates more pressure and reduces CSF flow around the optic nerve. This change may play a role in the development and progression of various ophthalmic and neurological diseases, including glaucoma.

Sodium and potassium intake in Hungarian children and adolescents: Comparison of two cross sectional studies

The purpose of the study was to assess the sodium and potassium intake among Hungarian children and adolescents. The study was undertaken to estimate daily sodium and potassium intake by measuring 24-h urinary electrolyte excretion rate in 200 Hungarian children (age 1–18 years). Daily sodium and potassium intake positively correlated with age, body mass index, and systolic blood pressure. As a result of the parallel trend and time course of urinary sodium and potassium excretion the sodium to potassium ratio, the important risk factor for essential hypertension remained practically unchanged with age. We did not find a significant difference in this ratio between hypertensive and normotensive, or obese and non-obese children. Sodium and potassium intake expressed per kilogram body weight decreased steadily with age, but there was no significant difference between boys and girls. When adjustment was made for age and body mass index, using multivariate regression analysis, the systolic blood pressure proved to be independent of urinary sodium excretion and daily potassium intake. Compared with Dietary Reference Intake values, the present study demonstrates that, on average, Hungarian children and adolescents have a diet rich in sodium, and poor in potassium, although during the last 20 years a favourable trend can be detected.

Stimulatory effects of human cytomegalovirus tegument protein pp71 lead to increased expression of CCL2 (monocyte chemotactic protein-1) during infection.

Human cytomegalovirus (CMV) is the most common infectious cause of congenital birth defects in developed countries. Studies of infected amniotic fluid and placentae show CMV infection leads to a pro-inflammatory shift in cytokine profiles with implications for pathogenesis of foetal disease. ELISA, immunofluorescence and real-time-PCR assays were used to investigate CCL2 (monocyte chemotactic protein-1) and TNF-α changes following CMV infection of human fibroblasts, as well as following transient expression of CMV gene products in HeLa cells. Infection of human fibroblasts with CMV AD169 resulted in increased cytoplasmic and extracellular expression of CCL2 during early stages of infection, followed by marked downregulation of the chemokine at late times. Induction of CCL2 was not observed with CMV clinical strain Merlin, consistent with the postulated immune-evasion potential of this genetically intact WT strain. Comparison between live and UV-irradiated virus infections showed that changes in CCL2 levels were a direct response to active CMV replication. There were no significant changes in TNF-α expression during a parallel time-course of CMV infection. In transient transfection assays, overexpression of CMV tegument protein pp71 resulted in intracellular and extracellular upregulation of CCL2 protein. mRNA analysis showed that pp71-induced elevation in CCL2 was mediated through transcriptional upregulation. The data showed that CMV-induced upregulation of CCL2 during early stages of infection was mediated, at least in part, by stimulation of viral pp71, which may contribute to viral pathogenesis through enhanced virus dissemination.

Influence of monkey dorsolateral prefrontal and posterior parietal activity on behavioral choice during attention tasks

The dorsolateral prefrontal and the posterior parietal cortex have both been implicated in the guidance of visual attention. Traditionally, posterior parietal cortex has been thought to guide visual bottom-up attention and prefrontal cortex to bias attention through top-down information. More recent studies suggest a parallel time course of activation of the two areas in bottom-up attention tasks, suggesting a common involvement, though these results do not necessarily imply identical roles. To address the specific roles of the two areas, we examined the influence of neuronal activity recorded from the prefrontal and parietal cortex of monkeys as they performed attention tasks based on choice probability and on correlation between reaction time and neuronal activity. The results revealed that posterior parietal but not dorsolateral prefrontal activity correlated with behavioral choice during the fixation period, prior to the appearance of the stimulus, resembling a bias factor. This preferential influence of posterior parietal activity on behavior was transient, so that dorsolateral prefrontal activity predicted choice after the appearance of the stimulus. Additionally, reaction time was better predicted by posterior parietal activity. These findings confirm the involvement of both dorsolateral prefrontal and posterior parietal cortex in the bottom-up guidance of visual attention, but indicate different roles of the two areas in the guidance of attention and a dynamic time course of their effects, influencing behavior at different stages of the task.

Immunoglobulin M, C-Reactive Protein and Complement Activation in Rat Hepatic Ischemia-Reperfusion Injury

Background: Ischemia-reperfusion (I/R) models have shown that C-reactive protein (CRP) and immunoglobulin M (IgM) are involved in complement activation. Binding of CRP and IgM to damaged cell membranes initiates complement activation and aggravates I/R injury in various organs. However, the time course of CRP- and IgM-mediated complement activation and the relation to hepatocellular injury and inflammation in liver I/R are unknown. Aim: To evaluate the time course of IgM- and CRP-related complement activation and the relation to hepatocellular injury and inflammation in a hepatic I/R rat model. Methods: Male Wistar rats were allocated to (1) five groups of animals exposed to 60 min of partial ischemia (70%) induced via clamping of the left segmental portal triad, followed by 0, 3, 6, 12 or 24 h of reperfusion (n = 6 in each group); (2) five groups of sham-operated animals with corresponding reperfusion times (n = 5), and (3) a control group sacrificed before ischemia (n = 5). Hepatocellular injury, inflammatory response, rat plasma CRP and IgM levels and immunohistochemical depositions of CRP, IgM and C3 were assessed for each group. Results: Histopathological injury scores of hematoxylin and eosin sections of ischemic liver lobes demonstrated increasing values throughout the reperfusion time with a peak at 12 h. Plasma aminotransferases (alanine aminotransferase and aspartate aminotransferase) significantly increased after 3 h of reperfusion, peaking at 6 h (3,100 ± 800 U/l; p < 0.05). Hepatic neutrophil influx significantly increased from 3 to 6 h of reperfusion (p < 0.05) and demonstrated the highest value at 12 h (1.1 ± 0.2 U/mg of protein). Plasma IL-6 levels in the ischemia groups showed peak values after 6 h of reperfusion, decreasing significantly thereafter (p < 0.05). Plasma CRP values reached highest levels after 3 h of reperfusion (mean 91 ± 5% of control pool), decreasing significantly thereafter. Rat IgM concentrations in plasma did not significantly change throughout the reperfusion time. Immunohistochemical depositions of IgM, CRP and C3 in ischemic lobes demonstrated a similar pattern in time, reaching maximum values at 12 h of reperfusion. The percentages of depositions of CRP and IgM were significantly correlated [r(S) = 0.569; p < 0.001; Spearman test]. The time course of C3 and CRP depositions throughout reperfusion and C3 and IgM staining were significantly similar [r(S) = 0.797 and r(S) = 0.656, respectively; p < 0.0001; ANOVA]. Conclusions: CRP and IgM depositions demonstrate a parallel time course throughout the reperfusion to hepatocellular damage, inflammatory response and activated complement deposition in this rat hepatic I/R model. Furthermore, the time course of CRP and IgM depositions was significantly similar to that of activated complement depositions.

Role of Akt signaling pathway in delayed cerebral vasospasm after subarachnoid hemorrhage in rats

Akt plays an important role in cell survival, proliferation, apoptosis and other activities. It also has been involved in maintaining smooth muscle cell contraction phenotypes in vitro and in vivo. Recent studies have focused on the inhibition of Akt in acute vasospasm and neuronal apoptosis after subarachnoid hemorrhage (SAH). However, its role in delayed cerebral vasospasm (DCVS) has not been reported. In this study, using a "two-hemorrhage" rat model of SAH, we examined the expression of p-Akt and the formation of vasospasm in the basilar arteries. To investigate the possible role of Akt in phenotypic switching, we performed immunohistochemical staining to examine expressions of SMα-actin and proliferating cell nuclear antigen (PCNA), markers of smooth muscle phenotypic switching. We found that the basilar arteries exhibited vasospasm after SAH and that vasospasm became most severe on day 7 after SAH. Elevated protein expression of p-Akt was detected 4days after SAH induction, peaked on day 7, and recovered on day 21, which was in a parallel time course to the development of DCVS. Moreover, results of immunohistochemical staining revealed enhanced expression of PCNA but gradual reduction in expression of SMα-actin from day 1 to day 7 after SAH; then, the expressions of PCNA and SMα-actin gradually recovered until day 21. These results support a novel mechanism in which the Akt signaling pathway plays an important role in the proliferation of smooth muscle cells (SMCs) rather than inducing phenotype switching in basilar arteries, which promotes the development of DCVS after SAH.

Nitrous Oxide Genotoxicity

Nitrous Oxide Genotoxicity

Expression and cell distribution of myeloid differentiation primary response protein 88 in the cerebral cortex following experimental subarachnoid hemorrhage in rats: A pilot study

Subarachnoid hemorrhage (SAH) which is mostly caused by aneurysm rupture causes a lot of death every year. Convincing evidence can be made that inflammation contributes to the poor outcome caused by SAH. Toll like receptors (TLRs), nuclear factor-kappaB (NF-κB), Interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α) are involved in the damaging inflammation process after SAH. Myeloid differentiation primary response protein 88 (MyD88) is essential to deliver TLRs signals down to NF-κB and pro-inflammatory factors. The study aims to detect the expression level of MyD88 and know more about the role of MyD88 after SAH. Sprague Dawley (SD) rats were randomly divided into sham group and SAH groups at 2h, 6h, 12h and on day 1, day 2, day 3, day 5 and day 7. SAH groups suffered experimental subarachnoid hemorrhage by injection of 0.3ml autoblood into the prechiasmatic cistern. MyD88 expression is measured by western blot analysis, real-time polymerase chain reaction (PCR), immunohistochemistry and immunofluorescence. The levels of TNF-α and IL-1β were measured by real-time PCR. Our results demonstrated MyD88 expression was increased after SAH, and peaked on day 1 and day 5, which showed a parallel time course to the up-regulation of IL-1β, there was a highly positive relationship between them. Immunohistochemistry and immunofluorescence results indicated up-regulated MyD88 was mainly located in neurons while over expressed MyD88 could also be found in astrocytes and microglia. These results might have important implications during the administration of specific MyD88 antagonists in order to prevent or reduce inflammatory response following SAH.

Potential contribution of SOCC to cerebral vasospasm after experimental subarachnoid hemorrhage in rats

Cerebral vasospasm (CVS) is the most treatable component of subarachnoid hemorrhage (SAH), which can be reduced by endothelin receptor antagonists. Endothelin-evoked vasospasm is considered to be mediated by Ca2+ influx in the smooth muscle through voltage-dependent Ca2+ channel (VDCC) and nonselective cation channels (NSCC). Because VDCC antagonists such as nimodipine have been shown to be relatively less effective than the endothelin receptor antagonists, it is assumed that NSCC maybe a more important component in mediating Ca2+ influx during CVS. In this study, we used the basilar arteries from a “two-hemorrhage” rat model of SAH to investigate expressions of transient receptor potential channel 1 (TRPC1), transient receptor potential channel 3 (TRPC3) and stromal interaction molecule 1 (STIM1), which are considered as the promising candidates constituting NSCC. To investigate the possible role of NSCC in phenotypic switching, we performed immunohistochemical staining to examine expressions of SMα-actin and PCNA, markers of smooth muscle phenotypic switching. We found that the basilar arteries exhibited vasospasm after SAH and that vasospasm became more severe on days 5 and 7 after SAH. Elevated mRNA and protein expressions of TRPC1 and STIM1 were detected after SAH and peaked on days 5 and 7, which was in a parallel time course to the development of cerebral vasospasm. The mRNA and protein expressions of TRPC3 were not changed in the SAH group when compared with those in the control. Results of immunohistochemical staining with anti-PCNA and anti-SMα-actin antibodies also showed enhanced expression of PCNA and disappearance of SMα-actin from day 1 to day 7. Taken together, the above results supported a novel mechanism that the components of store-operated calcium channels, TRPC1 and STIM1 mediated the Ca2+ influx and phenotypic switching in smooth muscle cells, which promoted the development of vasospasm after SAH. TRPC3, which is a component of receptor-operated calcium channels, was not involved in the above-mentioned mechanism.

Subunit-specific Incorporation Efficiency and Kinetics in Mitochondrial Complex I Homeostasis

Studies employing native PAGE suggest that most nDNA-encoded CI subunits form subassemblies before assembling into holo-CI. In addition, in vitro evidence suggests that some subunits can directly exchange in holo-CI. Presently, data on the kinetics of these two incorporation modes for individual CI subunits during CI maintenance are sparse. Here, we used inducible HEK293 cell lines stably expressing AcGFP1-tagged CI subunits and quantified the amount of tagged subunit in mitoplasts and holo-CI by non-native and native PAGE, respectively, to determine their CI incorporation efficiency. Analysis of time courses of induction revealed three subunit-specific patterns. A first pattern, represented by NDUFS1, showed overlapping time courses, indicating that imported subunits predominantly incorporate into holo-CI. A second pattern, represented by NDUFV1, consisted of parallel time courses, which were, however, not quantitatively overlapping, suggesting that imported subunits incorporate at similar rates into holo-CI and CI assembly intermediates. The third pattern, represented by NDUFS3 and NDUFA2, revealed a delayed incorporation into holo-CI, suggesting their prior appearance in CI assembly intermediates and/or as free monomers. Our analysis showed the same maximum incorporation into holo-CI for NDUFV1, NDUFV2, NDUFS1, NDUFS3, NDUFS4, NDUFA2, and NDUFA12 with nearly complete loss of endogenous subunit at 24 h of induction, indicative of an equimolar stoichiometry and unexpectedly rapid turnover. In conclusion, the results presented demonstrate that newly formed nDNA-encoded CI subunits rapidly incorporate into holo-CI in a subunit-specific manner.

Apicobasal gradient of left ventricular myocardial edema underlies transient T-wave inversion and QT interval prolongation (Wellens’ ECG pattern) in Tako-Tsubo cardiomyopathy

Tako-Tsubo cardiomyopathy (TTC) presents with chest pain, ST-segment elevation followed by T-wave inversion and QT interval prolongation (Wellens' electrocardiographic [ECG] pattern), and left ventricular dysfunction, which may mimic an acute coronary syndrome. To assess the pathophysiologic basis of the Wellens' ECG pattern in TTC by characterization of underlying myocardial changes by using cardiac magnetic resonance (CMR). The study population included 20 consecutive patients with TTC (95% women; mean age 65.3 ± 10.4 years) who underwent CMR studies both in the initial phase and after 3-month follow-up by using a protocol that included cine images, T2-weighted sequences for myocardial edema, and post-contrast sequences for late gadolinium enhancement. Quantitative ECG indices of repolarization, such as maximal amplitude of negative T waves, sum of the amplitudes of negative T waves, and maximum corrected QT interval (QTc max), were correlated to CMR findings. At the time of initial CMR study, there was a significant linear correlation between the apicobasal ratio of T2-weighted signal intensity for myocardial edema and the maximal amplitude of negative T waves (ρ = 0.498; P = .02), sum of the amplitudes of negative T waves (ρ = 0.483; P = .03), and maximum corrected QT interval (ρ = 0.520; P = .02). Repolarization indices were unrelated to either late gadolinium enhancement or quantitative cine parameters. Wellens' ECG abnormalities and myocardial edema showed a parallel time course of development and resolution on initial and follow-up CMR studies. Our study results show that the ischemic-like Wellens' ECG pattern in TTC coincides and quantitatively correlates with the apicobasal gradient of myocardial edema as evidenced by using CMR. Dynamic negative T waves and QTc prolongation are likely to reflect the edema-induced transient inhomogeneity and dispersion of repolarization between apical and basal left ventricular regions.

An in planta, Agrobacterium-mediated transient gene expression method for inducing gene silencing in rice (Oryza sativa L.) leaves

BackgroundLocalized introduction and transient expression of T-DNA constructs mediated by agro-infiltration of leaf tissues has been largely used in dicot plants for analyzing the transitivity and the cell-to cell movement of the RNAi signal. In cereals, however, the morphology of the leaf and particularly the structure of the leaf epidermis, prevent infiltration of a bacterial suspension in cells by simple pressure, a method otherwise successful in dicots leaves. This study aimed at establishing a rapid method for the functional analysis of rice genes based on the triggering of RNA interference (RNAi) following Agrobacterium-mediated transient transformation of leaves.ResultsUsing an agro-infection protocol combining a wound treatment and a surfactant, we were able to obtain in a reliable manner transient expression of a T-DNA-borne uidA gene in leaf cells of japonica and indica rice cultivars. Using this protocol to transiently inhibit gene expression in leaf cells, we introduced hairpin RNA (hpRNA) T-DNA constructs containing gene specific tags of the phytoene desaturase (OsPDS) and of the SLENDER 1 (OsSLR1) genes previously proven to trigger RNAi of target genes in stable transformants. SiRNA accumulation was observed in the agro-infected leaf area for both constructs indicating successful triggering of the silencing signal. Accumulation of secondary siRNA was observed in both stably and transiently transformed leaf tissues expressing the HpRNA OsSLR1 construct. Gene silencing signalling was investigated in monitoring the parallel time course of OsPDS-derived mRNA and siRNA accumulation in the agro-infiltrated leaf area and adjacent systemic sectors. The sensitive RT-Q-PCR method evidenced a consistent, parallel decrease of OsPDS transcripts in both the agroinfiltred and adjacent tissues, with a time lag for the latter.ConclusionsThese results indicate that the method is efficient at inducing gene silencing in the agro-infected leaf area. The transfer of low amounts of siRNA, probably occurring passively through the symplastic pathway from the agro-infected area, seemed sufficient to trigger degradation of target transcripts in the adjacent tissues. This method is therefore well suited to study the cell-to-cell movement of the silencing signal in a monocot plant and further test the functionality of natural and artificial miRNA expression constructs.Electronic supplementary materialThe online version of this article (doi:10.1186/1939-8433-5-23) contains supplementary material, which is available to authorized users.

194 Recombinant High-mobility Group Box 1 Protein (HMGB-1) Promotes Myeloid Differentiation Primary Response Protein 88 (Myd88) Upregulation in Mouse Primary Cortical Neurons

INTRODUCTION: Myeloid differentiation primary response protein 88 (Myd88) is a vital factor for inflammation and immunity, and high-mobility group box 1 protein (HMGB-1) is released after injury from neurons and may contribute to the initial stages of inflammatory response. Therefore, the current study was intended to investigate the expression of Myd88 in the cultured neurons following recombinant HMGB-1 addition and to clarify the potential role of Myd88 after neuron injury in vitro. METHODS: The cultured neurons were randomly divided into six groups: control group and HMGB-1 groups at hours 1, 6, 12, 24, and 48. The cultured neurons in HMGB-1 groups were subjected to addition of recombinant HMGB-1. The expression of Myd88 was assessed by quantitative real-time polymerase chain reaction (PCR), Western blotting and immunofluorescence, and nuclear factor kappa B (NF-kB) binding activity was detected by electrophoretic mobility shift assay (EMSA), and the levels of tumor necrosis factor-a (TNF-a) and Interleukin 1ß (IL-1ß) were measured by quantitative real-time PCR. RESULTS: The elevated mRNA and protein levels of Myd88, peaking at 24 h, were detected after HMGB-1 addition. NF-kB, TNF-a, and IL-1ß also ascended significantly after HMGB-1 addition. Interestingly, Myd88 increasingly expressed in a parallel time course to the upregulation of NF-kB, TNF-a, and IL-1ß. CONCLUSION: These findings indicated the possible role of Myd88 in the inflammatory response after neuron injury, and might provide an attractive therapeutic approach of targeting the Myd88 cascade to achieve better outcomes for patients after central nervous system injury.

Recombinant high-mobility group box 1 protein (HMGB-1) promotes myeloid differentiation primary response protein 88 (Myd88) upregulation in mouse primary cortical neurons

Myeloid differentiation primary response protein 88 (Myd88) is a vital factor for inflammation and immunity, and high-mobility group box 1 protein (HMGB-1) can be released from neurons after injury and may contribute to the initial stages of inflammatory response. Therefore, the current study was intended to investigate the expression of Myd88 in cultured neurons following recombinant HMGB-1 (rHMGB-1) addition and to clarify the potential role of Myd88 after neuron injury in vitro. The cultured neurons were randomly divided into six groups: control group and rHMGB-1 groups at hours 1, 6, 12, 24, and 48. The cultured neurons in rHMGB-1 groups were subjected to rHMGB-1 addition. The expression of Myd88 was assessed by quantitative real-time polymerase chain reaction (PCR), Western blotting and immunofluorescence, and nuclear factor kappa B (NF-κB) DNA-binding activity was detected by electrophoretic mobility shift assay, and the levels of tumor necrosis factor-α (TNF-α) and interleukin 1β (IL-1β) were measured by quantitative real-time PCR. The elevated mRNA and protein levels of Myd88, peaking at 24 h, were detected after rHMGB-1 addition. NF-κB, TNF-α, and IL-1β also ascended significantly after rHMGB-1 addition. Interestingly, Myd88 increasingly expressed in a parallel time course to the upregulation of NF-κB, TNF-α, and IL-1β. These findings indicated a possible role of Myd88 in the inflammatory response after neuron injury, and might provide an attractive therapeutic approach of targeting the Myd88 cascade to achieve better outcomes for patients with central nervous system injury.

Erythropoietin effect on sensorimotor recovery after contusive spinal cord injury: An electrophysiological study in rats

Spinal cord injury (SCI) is a debilitating clinical condition, characterized by a complex of neurological dysfunctions. It has been shown in rats that the acute administration of recombinant human erythropoietin (rhEPO) following a contusive SCI improves the recovery of hindlimb motor function, as measured with the locomotor BBB (Basso, Beattie, Bresnahan) scale. This scale evaluates overall locomotor activity, without testing whether the rhEPO-induced motor recovery is due to a parallel recovery of sensory and/or motor pathways. Aim of the present study was to utilize an electrophysiological test to evaluate, in a rat model of contusive SCI, the transmission of both ascending and descending pathways across the damaged cord at 2, 5, 7, 11, and 30days after lesion, in animals treated with rhEPO (n=25) vs saline solution (n=25). Motor potentials evoked by epicortical stimulation were recorded in the spinal cord, and sensory-evoked potentials evoked by spinal stimulation were recorded at the cortical level. In the same animals BBB score and immunocytochemical evaluation of the spinal segments caudal to the lesion were performed. In rhEPO-treated animals results show a better general improvement both in sensory and motor transmission through spared spinal pathways, supposedly via the reticulo-spinal system, with respect to saline controls. This improvement is most prominent at relatively early times. Overall these features show a parallel time course to the changes observed in BBB score, suggesting that EPO-mediated spared spinal cord pathways might contribute to the improvement in transmission which, in turn, might be responsible for the recovery of locomotor function.

Letter by Pérez de Prado et al Regarding Article, “Delayed Coverage in Malapposed and Side-Branch Struts With Respect to Well-Apposed Struts in Drug-Eluting Stents: In Vivo Assessment With Optical Coherence Tomography”

HomeCirculationVol. 125, No. 10Letter by Pérez de Prado et al Regarding Article, “Delayed Coverage in Malapposed and Side-Branch Struts With Respect to Well-Apposed Struts in Drug-Eluting Stents: In Vivo Assessment With Optical Coherence Tomography” Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBLetter by Pérez de Prado et al Regarding Article, “Delayed Coverage in Malapposed and Side-Branch Struts With Respect to Well-Apposed Struts in Drug-Eluting Stents: In Vivo Assessment With Optical Coherence Tomography” Armando Pérez de Prado, MD, Carlos Cuellas-Ramón, MD and Felipe Fernández-Vázquez, MD, PhD Armando Pérez de PradoArmando Pérez de Prado Search for more papers by this author , Carlos Cuellas-RamónCarlos Cuellas-Ramón Search for more papers by this author and Felipe Fernández-VázquezFelipe Fernández-Vázquez Search for more papers by this author Originally published13 Mar 2012https://doi.org/10.1161/CIRCULATIONAHA.111.064493Circulation. 2012;125:e458To the Editor:We read with great interest the article by Gutiérrez-Chico et al,1 which reports delayed coverage of incompletely apposed and nonapposed side-branch struts with respect to well-apposed struts in drug-eluting stents as assessed by optical coherence tomography. The relationship between incomplete stent apposition and delayed strut coverage has previously been explored. However, the study of fate of nonapposed side-branch struts is quite original.These struts, placed across the origin of side branches, have systematically been excluded from analysis in most of the published optical coherence tomography studies. Intuitively, they were deemed not suitable to be covered. These new results abrogate that theory, demonstrating by optical coherence tomography analysis that up to 78.1% of these struts appear covered 9 to 13 months after drug-eluting stent implantation. This finding is indeed relevant, but a question arises: What is covering those struts? As the authors acknowledge, optical coherence tomography covering is not a surrogate for neointimal healing. The vascular healing process involves the participation of the coagulation system and many types of cells in different phases, all of them covering the stent struts. Optical coherence tomography is unable to distinguish between fibrin, giant cells, granulomatous reaction, and degree of endothelialization.2 Until we can confirm recent findings with optical density analysis that claims being able to distinguish between neointima and fibrin/thrombus,3 we can only search for clues. Can the authors offer any extra information that helps in this endeavor? Is there any difference in the thickness of the coverage between apposed and nonapposed struts?We have previously reported that these nonapposed side-branch struts show a high reendothelialization rate in a swine coronary model.4 Our data coincide with the results presented by Gutiérrez-Chico et al1: Nonapposed side-branch struts show delayed endothelialization but a parallel time course to the rest of struts. The endothelial cells that cover these nonapposed struts necessarily come from circulating endothelial progenitor cells because there is no chance of repopulation from the surrounding area. The animal models use young, healthy individuals as opposed to human patients with defective healing mechanisms. Whether these findings predict a similar behavior in the clinical setting is still unknown.It is common to observe nonapposed struts placed over the side-branch origin; up to 41% of the analyzed stents were nonapposed.1 In daily practice, the rate should be even higher because the analysis refers to randomized studies with moderate stent lengths. Therefore, the clinical relevance of the status of these struts is not negligible. Whatever the coverage of these struts is, some issues must be considered. One of them emerges from the use of the new bioresorbable scaffolds. The struts of these prostheses, also those located over the side-branch takeoff, finally disappear as expected.5 Do they show a strut coverage similar to that observed in metallic stents? Can the breakdown of the scaffold cause clinically relevant side-branch embolism? We guess this topic will prompt further analyses of these floating struts in different situations.Armando Pérez de Prado, MDCarlos Cuellas-Ramón, MDFelipe Fernández-Vázquez, MD, PhD Fundación Investigación Sanitaria en León–HemoLeon Instituto de Biomedicina (IBIOMED) Hospital de León–Universidad de León León, SpainDisclosuresNone.