Anne Sebastiani

Expression of Na+-d-glucose cotransporter SGLT2 in rodents is kidney-specific and exhibits sex and species differences

With a novel antibody against the rat Na(+)-D-glucose cotransporter SGLT2 (rSGLT2-Ab), which does not cross-react with rSGLT1 or rSGLT3, the ∼75-kDa rSGLT2 protein was localized to the brush-border membrane (BBM) of the renal proximal tubule S1 and S2 segments (S1 > S2) with female-dominant expression in adult rats, whereas rSglt2 mRNA expression was similar in both sexes. Castration of adult males increased the abundance of rSGLT2 protein; this increase was further enhanced by estradiol and prevented by testosterone treatment. In the renal BBM vesicles, the rSGLT1-independent uptake of [(14)C]-α-methyl-D-glucopyranoside was similar in females and males, suggesting functional contribution of another Na(+)-D-glucose cotransporter to glucose reabsorption. Since immunoreactivity of rSGLT2-Ab could not be detected with certainty in rat extrarenal organs, the SGLT2 protein was immunocharacterized with the same antibody in wild-type (WT) mice, with SGLT2-deficient (Sglt2 knockout) mice as negative control. In WT mice, renal localization of mSGLT2 protein was similar to that in rats, whereas in extrarenal organs neither mSGLT2 protein nor mSglt2 mRNA expression was detected. At variance to the findings in rats, the abundance of mSGLT2 protein in the mouse kidneys was male dominant, whereas the expression of mSglt2 mRNA was female dominant. Our results indicate that in rodents the expression of SGLT2 is kidney-specific and point to distinct sex and species differences in SGLT2 protein expression that cannot be explained by differences in mRNA.

Single Administration of Tripeptide α-MSH(11–13) Attenuates Brain Damage by Reduced Inflammation and Apoptosis after Experimental Traumatic Brain Injury in Mice

Following traumatic brain injury (TBI) neuroinflammatory processes promote neuronal cell loss. Alpha-melanocyte-stimulating hormone (α-MSH) is a neuropeptide with immunomodulatory properties, which may offer neuroprotection. Due to short half-life and pigmentary side-effects of α-MSH, the C-terminal tripeptide α-MSH(11–13) may be an anti-inflammatory alternative. The present study investigated the mRNA concentrations of the precursor hormone proopiomelanocortin (POMC) and of melanocortin receptors 1 and 4 (MC1R/MC4R) in naive mice and 15 min, 6, 12, 24, and 48 h after controlled cortical impact (CCI). Regulation of POMC and MC4R expression did not change after trauma, while MC1R levels increased over time with a 3-fold maximum at 12 h compared to naive brain tissue. The effect of α-MSH(11–13) on secondary lesion volume determined in cresyl violet stained sections (intraperitoneal injection 30 min after insult of 1 mg/kg α-MSH(11–13) or 0.9% NaCl) showed a considerable smaller trauma in α-MSH(11–13) injected mice. The expression of the inflammatory markers TNF-α and IL-1β as well as the total amount of Iba-1 positive cells were not reduced. However, cell branch counting of Iba-1 positive cells revealed a reduced activation of microglia. Furthermore, tripeptide injection reduced neuronal apoptosis analyzed by cleaved caspase-3 and NeuN staining. Based on the results single α-MSH(11–13) administration offers a promising neuroprotective property by modulation of inflammation and prevention of apoptosis after traumatic brain injury.

Propofol impairs neurogenesis and neurologic recovery and increases mortality rate in adult rats after traumatic brain injury.

Objective:Limited data are available on the influence of sedation for critical care therapy with the widely used anesthetic propofol on recovery from acute traumatic brain injury. To establish the influence of propofol on endogenous neurogenesis and functional recovery after traumatic brain injury, rats were sedated with propofol either during or 2 hours after experimental traumatic brain injury. Design:Randomized controlled animal study. Setting:University research laboratory. Subjects:One hundred sixteen male Sprague Dawley rats. Interventions:Mechanical brain lesion by controlled cortical impact. Measurements and Main Results:This study investigated the dose-dependent influence of propofol (36 or 72 mg/kg/hr) either during controlled cortical impact induction or in a delayed application protocol 2 hours after experimental traumatic brain injury. Infusion of propofol resulted in 1) aggravation of neurologic dysfunction, 2) increased 28-day mortality rate, and 3) impaired posttraumatic neurogenesis (5-bromo-2-deoxyuridine + NeuN-positive cells). Application of propofol during trauma induction afforded a significant stronger effect in the high-dose group compared with low-dose propofol. In the posttrauma protocol, animals were sedated with sevoflurane during the controlled cortical impact injury, and propofol was given after an awake phase. In these animals, propofol increased mortality rate and impaired neurologic function and neurogenesis compared with animals without delayed propofol anesthesia. Conclusions:The results show that propofol may prevent or limit reparative processes in the early-phase postinjury. The results therefore indicate that anesthetics may be potentially harmful not only in very young mammalians but also in adult animals following acute cerebral injuries. The results provide first evidence for an altered sensitivity for anesthesia-related negative effects on neurogenesis, functional outcome, and survival in adult rats with brain lesions.

Traumatic brain injury results in rapid pericyte loss followed by reactive pericytosis in the cerebral cortex.

Accumulating evidence suggests a pivotal role of PDGFRß positive cells, a specific marker for central nervous system (CNS) pericytes, in tissue scarring. Identification of cells that contribute to tissue reorganization in the CNS upon injury is a crucial step to develop novel treatment strategies in regenerative medicine. It has been shown that pericytes contribute to scar formation in the spinal cord. It is further known that ischemia initially triggers pericyte loss in vivo, whilst brain trauma is capable of inducing pericyte detachment from cerebral vessels. These data point towards a significant role of pericytes in CNS injury. The temporal and spatial dynamics of PDGFRß cells and their responses in traumatic brain injury are poorly understood. Here we show that PDGFRß positive cells initially decline in the acute phase following experimental traumatic brain injury. However, PDGFRß positive cells increase significantly in the trauma zone days after brain injury. Using various pericyte markers we identify these cells to be pericytes that are demarcated by reactive gliosis. Our data indicate that brain trauma causes a biphasic response of pericytes in the early phase of brain trauma that may be of relevance for the understanding of pathological cellular responses in traumatic brain injury.

2-Methoxyestradiol confers neuroprotection and inhibits a maladaptive HIF-1α response after traumatic brain injury in mice.

HIF‐1α is pivotal for cellular homeostasis in response to cerebral ischemia. Pharmacological inhibition of HIF‐1α may reduce secondary brain damage by targeting post‐translational mechanisms associated with its proteasomal degradation and nuclear translocation. This study examined the neuroprotective effects of 2‐methoxyestradiol (2ME2), the involved HIF‐1α‐dependent response, and alternative splicing in exon 14 of HIF‐1α (HIF‐1α∆Ex14) after traumatic brain injury (TBI) in mice. Intraperitoneal 2ME2 administration 30 min after TBI caused a dose‐dependent reduction in secondary brain damage after 24 h. 2ME2 was physiologically tolerated, showed no effects on immune cell brain migration, and mitigated trauma‐induced brain expression of neuropathologically relevant HIF‐1α target genes encoding for Plasminogen activator inhibitor 1 and tumor necrosis factor alpha. Moreover, TBI‐induced expression of pro‐apoptotic BNIP3 was attenuated by 2ME2 treatment. Alternatively, spliced HIF‐1α∆Ex14 was substantially up‐regulated from 6 to 48 h after TBI. In vitro, nuclear location and gene transcription activity of HIF‐1α∆Ex14 were impaired compared to full‐length HIF‐1α, but no effects on nuclear translocation of the transcriptional complex partner HIF‐1β were observed. This study demonstrates that 2ME2 confers neuroprotection after TBI. While the role of alternatively spliced HIF‐1α∆Ex14 remains elusive, the in vivo data provide evidence that inhibition of a maladaptive HIF‐1α‐dependent response contributes to the neuroprotective effects of 2ME2.

Xenon Improves Neurologic Outcome and Reduces Secondary Injury Following Trauma in an In Vivo Model of Traumatic Brain Injury

Objectives:To determine the neuroprotective efficacy of the inert gas xenon following traumatic brain injury and to determine whether application of xenon has a clinically relevant therapeutic time window. Design:Controlled animal study. Setting:University research laboratory. Subjects:Male C57BL/6N mice (n = 196). Interventions:Seventy-five percent xenon, 50% xenon, or 30% xenon, with 25% oxygen (balance nitrogen) treatment following mechanical brain lesion by controlled cortical impact. Measurements and Main Results:Outcome following trauma was measured using 1) functional neurologic outcome score, 2) histological measurement of contusion volume, and 3) analysis of locomotor function and gait. Our study shows that xenon treatment improves outcome following traumatic brain injury. Neurologic outcome scores were significantly (p < 0.05) better in xenon-treated groups in the early phase (24 hr) and up to 4 days after injury. Contusion volume was significantly (p < 0.05) reduced in the xenon-treated groups. Xenon treatment significantly (p < 0.05) reduced contusion volume when xenon was given 15 minutes after injury or when treatment was delayed 1 or 3 hours after injury. Neurologic outcome was significantly (p < 0.05) improved when xenon treatment was given 15 minutes or 1 hour after injury. Improvements in locomotor function (p < 0.05) were observed in the xenon-treated group, 1 month after trauma. Conclusions:These results show for the first time that xenon improves neurologic outcome and reduces contusion volume following traumatic brain injury in mice. In this model, xenon application has a therapeutic time window of up to at least 3 hours. These findings support the idea that xenon may be of benefit as a neuroprotective treatment in patients with brain trauma.

Robotic assisted prostatic surgery in the Trendelenburg position does not impair cerebral oxygenation measured using two different monitors: A clinical observational study.

BACKGROUND Robotic assisted prostatic surgery is frequently used because of its reduced side-effects compared with conventional surgery. During surgery, an extreme Trendelenburg position and CO2 pneumoperitoneum are necessary, which may lead to cerebral oedema, can potentially reduce brain perfusion and therefore could impair cerebral oxygenation. Cerebral oxygen saturation can be measured non-invasively using near-infrared spectroscopy (NIRS). OBJECTIVE The hypothesis of the present study was that steep Trendelenburg positioning during robotic assisted prostatic surgery impairs cerebral oxygen saturation measured using two different NIRS monitors. DESIGN Clinical observational study. SETTING Primary care university hospital, study period from March 2012 to February 2013. PATIENTS A total of 29 patients scheduled for robotic assisted prostatic surgery in a steep Trendelenburg position. INTERVENTIONS Cerebral oxygen saturation was measured throughout anaesthesia using the INVOS sensor (a trend monitor using two infrared wavelengths) for one hemisphere and the FORE-SIGHT sensor (a monitor using four wavelengths of laser light to calculate absolute oxygen saturation) for the other hemisphere in an alternate randomisation. MAIN OUTCOME MEASURE Changes in cerebral oxygenation of more than 5% during surgery in the Trendelenburg position. RESULTS The median duration of Trendelenburg positioning was 190 (interquartile range 130 to 230) min. Cerebral oxygen saturation decreased with INVOS from 74 ± 5% at baseline to a lowest value of 70 ± 4% with a slope of −0.0129 min−1 (P <0.01) and with FORE-SIGHT from 72 ± 5% at baseline to a nadir of 70 ± 3% with a slope of −0.008 min−1 (P <0.01). Comparing INVOS with FORE-SIGHT, there was a good association, with a slope of 0.86 ± 0.04 (P <0.01). CONCLUSION Both monitors showed a clinically irrelevant decrease in cerebral oxygen saturation of less than 5% over 4 h in a steep Trendelenburg position combined with CO2 pneumoperitoneum in patients undergoing robotic assisted prostatic surgery. This extreme positioning seems to be acceptable with regard to cerebral oxygenation. TRIAL REGISTRATION clinicaltrials.gov Identifier: ID NCT01275898.

Proneurotrophin Binding to P75 Neurotrophin Receptor (P75ntr) Is Essential for Brain Lesion Formation and Functional Impairment after Experimental Traumatic Brain Injury.

Traumatic brain injury (TBI) initiates an excessive mediator release of e.g. neurotrophins, which promote neuronal survival, differentiation, and modulate synaptic plasticity. Paradoxically, mature forms of neurotrophins promote neuronal survival, whereas unprocessed forms of neurotrophins induce cell death through p75 neurotrophin receptor (p75NTR) signaling. p75NTR is widely expressed during synaptogenesis and is subsequently downregulated in adulthood. Repair mechanisms after acute cerebral insults can reactivate its expression. Therefore, the influence of p75NTR on secondary brain damage was addressed. mRNA levels of p75NTR and its ligands were quantified in brain tissue up to 7 days after experimental TBI (controlled cortical impact; CCI). Brain damage, motor function and inflammatory marker gene expression were determined in mice lacking the proneurotrophin-binding site of the p75NTR protein (NGFR(-/-)) and wild type littermates (NGFR(+/+)) 24 h and 5 days after CCI. In addition, the effect of TAT-Pep5 (pharmacological inhibitor of the intracellular p75NTR death domain) on lesion volume was evaluated 24 h after insult. p75NTR mRNA levels were induced nine-fold by TBI. In NGFR(-/-) mice, lesion volume was reduced by 29% at 24 h and by 21% 5 days after CCI. Motor coordination was significantly improved 24 h after trauma compared with the wild type. Pharmacological inhibition of the p75NTR signaling reduced lesion volume by 18%. The present study presents first time evidence that genetic mutation of the neurotrophin interaction site of p75NTR strongly limits post-traumatic cell death. In addition, we revealed pharmacological targeting of the intracellular p75NTR cell death domain as a promising approach to limit acute brain damage.

Posttraumatic Propofol Neurotoxicity Is Mediated via the Pro-Brain-Derived Neurotrophic Factor-p75 Neurotrophin Receptor Pathway in Adult Mice.

Objectives:The gamma-aminobutyric acid modulator propofol induces neuronal cell death in healthy immature brains by unbalancing neurotrophin homeostasis via p75 neurotrophin receptor signaling. In adulthood, p75 neurotrophin receptor becomes down-regulated and propofol loses its neurotoxic effect. However, acute brain lesions, such as traumatic brain injury, reactivate developmental-like programs and increase p75 neurotrophin receptor expression, probably to foster reparative processes, which in turn could render the brain sensitive to propofol-mediated neurotoxicity. This study investigates the influence of delayed single-bolus propofol applications at the peak of p75 neurotrophin receptor expression after experimental traumatic brain injury in adult mice. Design:Randomized laboratory animal study. Setting:University research laboratory. Subjects:Adult C57BL/6N and nerve growth factor receptor–deficient mice. Interventions:Sedation by IV propofol bolus application delayed after controlled cortical impact injury. Measurements and Main Results:Propofol sedation at 24 hours after traumatic brain injury increased lesion volume, enhanced calpain-induced &agr;II-spectrin cleavage, and increased cell death in perilesional tissue. Thirty-day postinjury motor function determined by CatWalk (Noldus Information Technology, Wageningen, The Netherlands) gait analysis was significantly impaired in propofol-sedated animals. Propofol enhanced pro–brain-derived neurotrophic factor/brain-derived neurotrophic factor ratio, which aggravates p75 neurotrophin receptor–mediated cell death. Propofol toxicity was abolished both by pharmacologic inhibition of the cell death domain of the p75 neurotrophin receptor (TAT-Pep5) and in mice lacking the extracellular neurotrophin binding site of p75 neurotrophin receptor. Conclusions:This study provides first evidence that propofol sedation after acute brain lesions can have a deleterious impact and implicates a role for the pro–brain-derived neurotrophic factor-p75 neurotrophin receptor pathway. This observation is important as sedation with propofol and other compounds with GABA receptor activity are frequently used in patients with acute brain pathologies to facilitate sedation or surgical and interventional procedures.

Comparison of speed-vacuum method and heat-drying method to measure brain water content of small brain samples

BACKGROUND A reliable measurement of brain water content (wet-to-dry ratio) is an important prerequisite for conducting research on mechanisms of brain edema formation. The conventionally used oven-drying method suffers from several limitations, especially in small samples. A technically demanding and time-consuming alternative is freeze-drying. NEW METHOD Centrifugal vacuum concentrators (e.g. SpeedVac/speed-vacuum drying) are a combination of vacuum-drying and centrifugation, used to reduce the boiling temperature. These concentrators have the key advantages of improving the freeze-drying speed and maintaining the integrity of dried samples, thus, allowing e.g. DNA analyses. In the present study, we compared the heat-oven with speed-vacuum technique with regard to efficacy to remove moisture from water and brain samples and their effectiveness to distinguish treatment paradigms after experimental traumatic brain injury (TBI) caused by controlled cortical impact (CCI). RESULTS Both techniques effectively removed water, the oven technique taking 24h and vacuum-drying taking 48h. Vacuum-drying showed lower variations in small samples (30-45mg) and was suitable for genomic analysis as exemplified by sex genotyping. The effect of sodium bicarbonate (NaBic8.4%) on brain edema formation after CCI was investigated in small samples (2×1mm). Only vacuum-drying showed low variation and significant improvement under NaBic8.4% treatment. COMPARISON WITH AN EXISTING METHOD The receiver operating curves (ROC) analysis demonstrated that vacuum-drying (area under the curve (AUC):0.867-0.967) was superior to the conventional heat-drying method (AUC:0.367-0.567). CONCLUSIONS The vacuum method is superior in terms of quantifying water content in small samples. In addition, vacuum-dried samples can also be used for subsequent analyses, e.g., PCR analysis.