Minocycline but not tigecycline is neuroprotective and reduces the neuroinflammatory response induced by the superimposition of sepsis upon traumatic brain injury.

OBJECTIVE: The development of sepsis in patients with traumatic brain injury increases mortality, exacerbates morphological and functional cerebral damage, and causes persistent neuroinflammation, including microglial activation. The administration of antibiotics possessing both antimicrobial and immunomodulatory activity might attenuate both sepsis and posttraumatic cerebral inflammation. We compared the potential therapeutic efficacy of two tetracyclines, minocycline and the newer generation tigecycline, on functional neurobehavioral impairment and regional histopathological damage in an experimental model of combined traumatic brain injury and sepsis. DESIGN: Prospective, experimental animal study. SETTING: University Research Laboratory. SUBJECTS: Adult male Sprague-Dawley rats. INTERVENTIONS: Controlled cortical impact was used to induce traumatic brain injury and cecal ligation and puncture for sepsis. Immediately following injury, animals were treated with minocycline (45 mg/kg intraperitoneal), tigecycline (7.5 mg/kg intraperitoneal), or saline every 12 hours for 3 days. MEASUREMENTS AND MAIN RESULTS: The development of sepsis and cerebral inflammatory response were evaluated, respectively, by 1) growth of peritoneal microorganisms and clinical variables and 2) tumor necrosis factor-α expression in the perilesional cortex. To assess posttraumatic outcome, vestibulomotor and cognitive function were evaluated at different time points for 14 days post injury whereupon animals were killed and cerebral tissue analyzed for lesion volume, regional hippocampal (CA1/CA3) cell death, and microglial activation in the perilesional cortex, lesion core zone, and choroid plexus. Treatment with both antibiotics reduced microorganism growth, body weight loss, and mortality but had no effect on vestibulomotor or cognitive function. Minocycline alone attenuated postinjury cortical lesion volume, hippocampal CA3 neuronal cell loss, tumor necrosis factor-α expression, and the extent of microglial activation and infiltration. CONCLUSIONS: The significantly heightened mortality caused by the superimposition of sepsis upon traumatic brain injury can be reduced by administration of both antibiotics but only minocycline can decrease the extent of cell death in selectively cortical and hippocampal brain regions, via, in part, a reduction in cerebral inflammation.

Combined effects of melatonin and all-trans retinoic acid and somatostatin on breast cancer cell proliferation and death: molecular basis for the anticancer effect of these molecules.

Melatonin has been shown to inhibit breast cancer cell growth in numerous studies. However, our understanding of the therapeutic effects of this hormone is still marginal and there is little information concerning its combination with other antitumor agents to achieve additional potential benefits. All-trans retinoic acids or somatostatin have been used in combination with melatonin in several pre-clinical and clinical trials, but they have never been combined altogether as an anti-breast cancer treatment. In the present study, we investigated whether the association of melatonin, all-trans retinoic acid and somatostatin leads to an enhanced anticancer activity in MCF-7 breast cancer cells. In such conditions, MCF-7 cells were investigated for cell growth/viability and proliferation, as well as for the expression of cyclin A, and components of the Notch and EGFR pathways, by Western blotting and confocal immunofluorescence. Electrophysiological, morphological, and biochemical analysis were also performed to reveal signs of cell damage and death. We found that melatonin in combination with all-trans retinoic acid and somatostatin potentiated the effects of melatonin alone on MCF-7 cell viability and growth inhibition; this phenomenon was associated with altered conductance through Ca²âº and voltage-activated K⁺ (BK) channels, and with substantial impairments of Notch-1 and epidermal growth factor (EGF)-mediated signaling. The combined treatment also caused a marked reduction in mitochondrial membrane potential and intracellular ATP production as well as induction of necrotic cell death. Taken together our results indicate that co-administration of melatonin with all-trans retinoic acid and somatostatin may be of significant therapeutic benefit in breast cancer.

Sepsis induces albuminuria and alterations in the glomerular filtration barrier: a morphofunctional study in the rat.

INTRODUCTION: Increased vascular permeability represents one of the hallmarks of sepsis. In the kidney, vascular permeability is strictly regulated by the 'glomerular filtration barrier' (GFB), which is comprised of glomerular endothelium, podocytes, their interposed basement membranes and the associated glycocalyx. Although it is likely that the GFB and its glycocalyx are altered during sepsis, no study has specifically addressed this issue. The aim of this study was to evaluate whether albuminuria--the hallmark of GFB perm-selectivity--occurs in the initial stage of sepsis and whether it is associated with morphological and biochemical changes of the GFB. METHODS: Cecal ligation and puncture (CLP) was used to induce sepsis in the rat. Tumor necrosis factor (TNF)-alpha levels in plasma and growth of microorganisms in the peritoneal fluid were evaluated at 0, 3 and 7 hours after CLP or sham-operation. At the same times, kidney specimens were collected and structural and ultrastructural alterations in the GFB were assessed. In addition, several components of GFB-associated glycocalyx, syndecan-1, hyluronan (HA) and sialic acids were evaluated by immunofluorescence, immunohistochemistry and lectin histochemistry techniques. Serum creatinine and creatinine clearance were measured to assess kidney function and albuminuria for changes in GFB permeability. Analysis of variance followed by Tukey's multiple comparison test was used. RESULTS: Septic rats showed increased TNF-alpha levels and growth of microorganisms in the peritoneal fluid. Only a few renal corpuscles had major ultrastructural and structural alterations and no change in serum creatinine or creatinine clearance was observed. Contrarily, urinary albumin significantly increased after CLP and was associated with diffuse alteration in the glycocalyx of the GFB, which consisted in a decrease in syndecan-1 expression and in HA and sialic acids contents. Sialic acids were also changed in their structure, exhibiting a higher degree of acetylation. CONCLUSIONS: In its initial phase, sepsis is associated with a significant alteration in the composition of the GFB-associated glycocalyx, with loss of GFB perm-selectivity as documented by albumin leakage into urine.

In vitro evaluation of the effects of low-intensity Nd:YAG laser irradiation on the inflammatory reaction elicited by bacterial lipopolysaccharide adherent to titanium dental implants.

BACKGROUND: The bacterial endotoxin lipopolysaccharide (LPS) represents a prime pathogenic factor of peri-implantitis because of its ability to adhere tenaciously to dental titanium implants. Despite this, the current therapeutic approach to this disease remains based mainly on bacterial decontamination, paying little attention to the neutralization of bioactive bacterial products. The purpose of the present study was to evaluate whether irradiation with low-energy neodymium-doped:yttrium, aluminum, and garnet (Nd:YAG) laser, in addition to the effects on bacterial implant decontamination, was capable of attenuating the LPS-induced inflammatory response. METHODS: RAW 264.7 macrophages or human umbilical vein endothelial cells were cultured on titanium disks coated with Porphyromonas gingivalis LPS, subjected or not to irradiation with the Nd:YAG laser, and examined for the production of inflammatory cytokines and the expression of morphologic and molecular markers of cell activation. RESULTS: Laser irradiation of LPS-coated titanium disks significantly reduced LPS-induced nitric oxide production and cell activation by the macrophages and strongly attenuated intercellular adhesion molecule-1 and vascular cell adhesion molecule expression, as well as interleukin-8 production by the endothelial cells. CONCLUSION: By blunting the LPS-induced inflammatory response, Nd:YAG laser irradiation may be viewed as a promising tool for the therapeutic management of peri-implantitis.

Skeletal myoblasts overexpressing relaxin improve differentiation and communication of primary murine cardiomyocyte cell cultures.

The possibility that resident myocardial progenitor cells may be re-activated by transplantation of exogenous stem cells into the post-infarcted heart has been suggested as a possible mechanism to explain the heart's functional improvement after stem cell therapy. Here we studied whether differentiation of mouse neonatal immature cardiomyocytes in vitro was influenced by mouse skeletal myoblasts C2C12, wild type or engineered to secrete the cardiotropic hormone relaxin. The cultured cardiomyocytes formed spontaneously beating clusters and temporally exhibited cardiac immunophenotypical (cKit, atrial natriuretic peptide, troponin T, connexin-43, HCN4) and electrical features (inward voltage-dependent Na(+), T- and L-type Ca(2+) currents, outward and inward K(+) currents, I(f) pacemaker current). These clusters were functionally connected through nanotubular structures and undifferentiated cardiac cells in the form of flattened stripes, bridging the clusters through connexin-43-containing gap junctions. These findings suggested the existence of long distance cell-to-cell communications among the cardiomyocyte aggregates involved in the intercellular transfer of Ca(2+) signals and organelles, likely required for coordination of myocardial differentiation. Co-presence of the myoblasts greatly increased cardiomyocyte differentiation and the amount of intercellular connections. In fact, these cells formed a structural support guiding elongation of nanotubules and stripe-like cells. The secretion of relaxin by the engineered myoblasts accelerated and enhanced the cardiomyogenic potential of the co-culture. These findings underscore the possibility that grafted myoblasts and cardiotropic factors, such as relaxin, may influence regeneration of resident immature cardiac cells, thus adding a tile to the mosaic of mechanisms involved in the functional benefits of cell transplantation for cardiac repair.

Systemic sepsis exacerbates mild post-traumatic brain injury in the rat.

The development of sepsis in patient suffering from traumatic brain injury (TBI) represents a frequent complication that has been associated with worsened global and neurological outcome. In an effort to better characterize the influence of sepsis following TBI, we developed an in vivo model of combined TBI and sepsis in the rat by coupling two validated models: (1) Controlled Cortical Impact (CCI) and (2) Cecal Ligation and Puncture (CLP). Possible contributing effects of sepsis on post-traumatic outcome were evaluated as mortality rate, body weight change, neurological motor (beam balance), cognitive (Morris water maze [MWM] for memory and learning) function, histopathological damage (lesion volume, cell counts in the CA1 and CA3 hippocampal areas), and morphological indices of inflammation (activated microglia and astrocytes) for the 14-day study period. In this study, we produced a mild TBI characterized by a low mortality rate, a transient delay in weight gain, and a transient impairment in motor and cognitive functions. The histological counterpart was represented by a cortical lesion in the area of impact at 14 days post-injury, associated with cell loss in the CA1 and CA3 hippocampal regions, and scarce infiltration of microglia. The superimposition of sepsis on this mild TBI model resulted in worsening of post-injury mortality and weight loss, significant exacerbation of post-injury motor deficit and cognitive impairments, and further exacerbation of neuronal cell death in the CA3 area together with over-expression and activation of microglial cells in the peri-lesional area. Altogether, our findings indicate that sepsis, when superimposed on TBI, exerts a negative effect on the evolution of post-traumatic damage.

Carbamylated erythropoietin is neuroprotective in an experimental model of traumatic brain injury.

OBJECTIVE: The well-documented neuroprotective effects of recombinant human erythropoietin (rhEPO) are commonly associated with untoward erythrocyte-stimulating effects (polycythemia), with subsequent risk of thromboembolic complications. A carbamylated-rhEPO (CEPO) derivative, which is neuroprotective but lacks hematopoietic activity, has been recently developed. In this study, we evaluated the neuroprotective capability of CEPO in an in vitro model of cerebral trauma in which rhEPO was previously shown to reduce posttraumatic cell death. DESIGN: Prospective, controlled experiment. SETTING: Animal, basic science laboratory. SUBJECTS: Wistar rats, 8 days old. INTERVENTIONS: Organotypic hippocampal slices, obtained from rat brains, were subjected to a well-characterized model of mechanical injury followed by addition of 10 IU/mL rhEPO, 10-100 IU/mL CEPO, or vehicle (injured control) to the incubation medium at different times to assess the temporal window of therapeutic neuroprotection. MEASUREMENTS AND MAIN RESULTS: Posttraumatic cell death was quantified at 12, 24, or 48 hrs after injury by measuring propidium iodide fluorescence in the selectively vulnerable CA1 hippocampal area. Posttraumatic injury, observed in injured, vehicle-treated hippocampal slices, was significantly attenuated by addition of either 10 IU/mL rhEPO or 10 IU/mL CEPO. The neuroprotective efficacy of 10 IU/mL rhEPO or CEPO remained intact even when administration was delayed 1 hr after trauma. Qualitative microscopy in semithin sections showed that both rhEPO and CEPO exerted a marked pyramidal neuron-sparing effect. CONCLUSION: Our study shows that 10 IU/mL CEPO exerts neuroprotective effects comparable with those of rhEPO in an in vitro model of mechanical cerebral trauma. Because CEPO lacks hematopoietic effects and seems to possess a prolonged therapeutic time window, this erythropoietin derivative may represent an exciting new pharmacologic tool in treating patients with mechanical injury to the brain.

Paracrine effects of transplanted myoblasts and relaxin on post-infarction heart remodelling.

In the post-infarcted heart, grafting of precursor cells may partially restore heart function but the improvement is modest and the mechanisms involved remain to be elucidated. Here, we explored this issue by transplanting C2C12 myoblasts, genetically engineered to express enhanced green fluorescent protein (eGFP) or eGFP and the cardiotropic hormone relaxin (RLX) through coronary venous route to swine with experimental chronic myocardial infarction. The rationale was to deliver constant, biologically effective levels of RLX at the site of cell engraftment. One month after engraftment, histological analysis showed that C2C12 myoblasts selectively settled in the ischaemic scar and were located around blood vessels showing an activated endothelium (ICAM-1-,VCAM-positive). C2C12 myoblasts did not trans-differentiate towards a cardiac phenotype, but did induce extracellular matrix remodelling by the secretion of matrix metalloproteases (MMP) and increase microvessel density through the expression of vascular endothelial growth factor (VEGF). Relaxin-producing C2C12 myoblasts displayed greater efficacy to engraft the post-ischaemic scar and to induce extracellular matrix re-modelling and angiogenesis as compared with the control cells. By echocardiography, C2C12-engrafted swine showed improved heart contractility compared with the ungrafted controls, especially those producing RLX. We suggest that the beneficial effects of myoblast grafting on cardiac function are primarily dependent on the paracrine effects of transplanted cells on extracellular matrix remodelling and vascularization. The combined treatment with myoblast transplantation and local RLX production may be helpful in preventing deleterious cardiac remodelling and may hold therapeutic possibility for post-infarcted patients.