Helena R. Watts

Dexmedetomidine provides renoprotection against ischemia-reperfusion injury in mice

IntroductionAcute kidney injury following surgery incurs significant mortality with no proven preventative therapy. We investigated whether the α2 adrenoceptor agonist dexmedetomidine (Dex) provides protection against ischemia-reperfusion induced kidney injury in vitro and in vivo.MethodsIn vitro, a stabilised cell line of human kidney proximal tubular cells (HK2) was exposed to culture medium deprived of oxygen and glucose. Dex decreased HK2 cell death in a dose-dependent manner, an effect attenuated by the α2 adrenoceptor antagonist atipamezole, and likely transduced by phosphatidylinositol 3-kinase (PI3K-Akt) signaling. In vivo C57BL/6J mice received Dex (25 μg/kg, intraperitoneal (i.p.)) 30 minutes before or after either bilateral renal pedicle clamping for 25 minutes or right renal pedicle clamping for 40 minutes and left nephrectomy.ResultsPre- or post-treatment with Dex provided cytoprotection, improved tubular architecture and function following renal ischemia. Consistent with this cytoprotection, dexmedetomidine reduced plasma high-mobility group protein B1 (HMGB-1) elevation when given prior to or after kidney ischemia-reperfusion; pretreatment also decreased toll-like receptor 4 (TLR4) expression in tubular cells. Dex treatment provided long-term functional renoprotection, and even increased survival following nephrectomy.ConclusionsOur data suggest that Dex likely activates cell survival signal pAKT via α2 adrenoceptors to reduce cell death and HMGB1 release and subsequently inhibits TLR4 signaling to provide reno-protection.

Isoflurane, a Commonly Used Volatile Anesthetic, Enhances Renal Cancer Growth and Malignant Potential via the Hypoxia-inducible Factor Cellular Signaling Pathway In Vitro

Background:Growing evidence indicates that perioperative factors, including choice of anesthetic, affect cancer recurrence after surgery although little is known about the effect of anesthetics on cancer cells themselves. Certain anesthetics are known to affect hypoxia cell signaling mechanisms in healthy cells by up-regulating hypoxia-inducible factors (HIFs). HIFs are also heavily implicated in tumorigenesis and high levels correlate with poor prognosis. Methods:Renal cell carcinoma (RCC4) cells were exposed to isoflurane for 2 h at various concentrations (0.5–2%). HIF-1&agr;, HIF-2&agr;, phospho-Akt, and vascular endothelial growth factor A levels were measured by immunoblotting at various time points (0–24 h). Cell migration was measured across various components of extracellular matrix, and immunocytochemistry was used to analyze proliferation rate and cytoskeletal changes. Results:Isoflurane up-regulated levels of HIF-1&agr; and HIF-2&agr; and intensified expression of vascular endothelial growth factor A. Exposed cultures contained significantly more cells (1.81 ± 0.25 vs. 1.00 of control; P = 0.03) and actively proliferating cells (89.4 ± 2.80 vs. 64.74 ± 7.09% of control; P = 0.016) than controls. These effects were abrogated when cells were pretreated with the Akt inhibitor, LY294002. Exposed cells also exhibited greater migration on tissue culture–coated (F = 16.89; P = 0.0008), collagen-coated (F = 20.99; P = 0.0003), and fibronectin-coated wells (F = 8.21; P = 0.011) as along with dramatic cytoskeletal rearrangement, with changes to both filamentous actin and &agr;-tubulin. Conclusions:These results provide evidence that a frequently used anesthetic can exert a protumorigenic effect on a human cancer cell line. This may represent an important contributory factor to high recurrence rates observed after surgery.

Xenon treatment protects against cold ischemia associated delayed graft function and prolongs graft survival in rats.

Prolonged hypothermic storage causes ischemia‐reperfusion injury (IRI) in the renal graft, which is considered to contribute to the occurrence of the delayed graft function (DGF) and chronic graft failure. Strategies are required to protect the graft and to prolong renal graft survival. We demonstrated that xenon exposure to human proximal tubular cells (HK‐2) led to activation of range of protective proteins. Xenon treatment prior to or after hypothermia–hypoxia challenge stabilized the HK‐2 cellular structure, diminished cytoplasmic translocation of high‐mobility group box (HMGB) 1 and suppressed NF‐κB activation. In the syngeneic Lewis‐to‐Lewis rat model of kidney transplantation, xenon exposure to donors before graft retrieval or to recipients after engraftment decreased caspase‐3 expression, localized HMGB‐1 within nuclei and prevented TLR‐4/NF‐κB activation in tubular cells; serum pro‐inflammatory cytokines IL‐1β, IL‐6 and TNF‐α were reduced and renal function was preserved. Xenon treatment of graft donors or of recipients prolonged renal graft survival following IRI in both Lewis‐to‐Lewis isografts and Fischer‐to‐Lewis allografts. Xenon induced cell survival or graft functional recovery was abolished by HIF‐1α siRNA. Our data suggest that xenon treatment attenuates DGF and enhances graft survival. This approach could be translated into clinical practice leading to a considerable improvement in long‐term graft survival.

The therapeutic potential of atorvastatin in a mouse model of postoperative cognitive decline.

Objective:Postoperative cognitive decline is emerging as a significant complication of surgery among older adults. Animal models indicate a central role of hippocampal inflammatory responses in the pathophysiology of postoperative cognitive decline. We hypothesized that atorvastatin, shown to exert neuroprotective potential in central nervous system (CNS) disorders, would attenuate neuroinflammation and improve cognitive function in mice after surgery and anesthesia. Methods:C57BL6 adult mice were pretreated with atorvastatin (250 &mgr;g) or vehicle, orally, for 5 days before undergoing unilateral nephrectomy under isoflurane anesthesia. We evaluated behavioral parameters related to cognitive function (fear conditioning and Morris Water Maze) and determined systemic and hippocampal interleukin-1&bgr; levels, postoperatively. Endothelial COX-2 expression, gross NF-&kgr;B and microglial (IBA1, CD68) activation, synaptic function (synapsin-1, PSD95, COX-2), heme oxygenase-1, and GSK3&bgr; were also examined. Results:Surgery induced a significant reduction in hippocampal-dependent fear response that was attenuated by treatment with atorvastatin, which also preserved spatial memory on day 7 after surgery. Atorvastatin evoked significant protection from hippocampal interleukin-1&bgr; production, but not systemic interleukin-1&bgr; production, accompanied by a marked reduction in hippocampal endothelial COX-2, NF-&kgr;B activation and decreased microglial reactivity. Surgery triggered an acute decline in synapsin-1, paralleled by an increase in postsynaptic COX-2 that was partially attenuated by atorvastatin. Furthermore, phosphorylation and inactivation of neuronal GSK3&bgr; was significantly enhanced after atorvastatin treatment. Conclusions:These findings indicate that cognitive decline is very likely associated with synaptic pathology after systemic and central inflammation induced by peripheral surgery/isoflurane anesthesia and suggest that the anti-inflammatory and neuroprotective properties of atorvastatin provide a rationale for its use as a therapeutic strategy for postoperative cognitive decline.

The Impact of Surgery and Anesthesia on Post-Operative Cognitive Decline and Alzheimer's Disease Development: Biomarkers and Preventive Strategies

Alzheimers disease (AD) is a major social and clinical burden in the elderly, affecting 5% of people aged over 65 and 20% aged over 80. Despite improved management, a cure has not been found and hence analysis of predisposing factors to identify preventive strategies has become increasingly important. Surgery and anesthesia have been proposed to increase the incidence of post-operative cognitive decline (POCD) and AD. This is hypothesized to be the result of a malignant neuroinflammatory response and subsequent synaptic impairment in the elderly and susceptible individuals. As a result, strategies are being explored to prevent surgery and anesthesia induced cognitive impairment. Whereas previously the diagnosis of AD was primarily dependent on clinical examination, biomarkers such as inflammatory cytokines, amyloid-β, and tau deposition in the cerebrospinal fluid have received increased attention. Nonetheless, AD is currently still treated symptomatically with acetylcholinesterase inhibitors and NMDA antagonists to improve cholinergic transmission and prevent glutamatergic excitotoxicity. Therapeutic success is, however, often not achieved, since these treatment methods do not address the ongoing neuroinflammatory processes and hence novel therapeutic and protective strategies are urgently needed. This review provides an insight into the current understanding of age-related cognitive impairment post-surgery and reflects on novel markers of AD pathogeneses exploring their use as targets for treatment. It gives a summary of recent efforts in preventing and treating POCD or AD with regards to the choice and depth of anesthesia, surgical strategy, and peri-operative medication, and discusses the mechanism of action and therapeutic prospects of novel agents.

Xenon Treatment Protects against Remote Lung Injury after Kidney Transplantation in Rats

Background:Ischemia–reperfusion injury (IRI) of renal grafts may cause remote organ injury including lungs. The authors aimed to evaluate the protective effect of xenon exposure against remote lung injury due to renal graft IRI in a rat renal transplantation model. Methods:For in vitro studies, human lung epithelial cell A549 was challenged with H2O2, tumor necrosis factor-&agr;, or conditioned medium from human kidney proximal tubular cells (HK-2) after hypothermia–hypoxia insults. For in vivo studies, the Lewis renal graft was stored in 4°C Soltran preserving solution for 24 h and transplanted into the Lewis recipient, and the lungs were harvested 24 h after grafting. Cultured lung cells or the recipient after engraftment was exposed to 70% Xe or N2. Phospho (p)-mammalian target of rapamycin (mTOR), hypoxia-inducible factor-1&agr; (HIF-1&agr;), Bcl-2, high-mobility group protein-1 (HMGB-1), TLR-4, and nuclear factor &kgr;B (NF-&kgr;B) expression, lung inflammation, and cell injuries were assessed. Results:Recipients receiving ischemic renal grafts developed pulmonary injury. Xenon treatment enhanced HIF-1&agr;, which attenuated HMGB-1 translocation and NF-&kgr;B activation in A549 cells with oxidative and inflammatory stress. Xenon treatment enhanced p-mTOR, HIF-1&agr;, and Bcl-2 expression and, in turn, promoted cell proliferation in the lung. Upon grafting, HMGB-1 translocation from lung epithelial nuclei was reduced; the TLR-4/NF-&kgr;B pathway was suppressed by xenon treatment; and subsequent tissue injury score (nitrogen vs. xenon: 26 ± 1.8 vs. 10.7 ± 2.6; n = 6) was significantly reduced. Conclusion:Xenon treatment confers protection against distant lung injury triggered by renal graft IRI, which is likely through the activation of mTOR-HIF-1&agr; pathway and suppression of the HMGB-1 translocation from nuclei to cytoplasm.

Differential effects of interleukin-1β and S100B on amyloid precursor protein in rat retinal neurons

Purpose: Interleukin-1β (IL-1β) and S100B calcium binding protein B (S100B) have been implicated in the pathogenesis of Alzheimer’s disease. Both are present in and around senile plaques and have been shown to increase levels of amyloid precursor protein (APP) mRNA in vitro. However, it is not known how either of these substances affects APP in vivo. Methods: We have studied the effects of IL-1β and S100B on the expression and processing of APP using a retinal-vitreal model. We have also investigated the effect of amyloid beta peptide (Aβ) on APP in the same system and the regulation of S100B production by Aβ and IL-1β from retinal glial cells. Results: Retinal ganglion cells constitutively express APP. However, after intravitreal injection of IL-1β or Aβ there was a marked reduction in APP levels as detected by Western blotting and IL-1β produced a decrease in APP immunoreactivity (IR). Nissl staining showed that the integrity of the injected retinas was unchanged after injection. Two days after S100B injection, there was a small reduction in APP-IR but this was accompanied by the appearance of some intensely stained large ganglion cells and there was some up-regulation in APP holoprotein levels on Western blot. Seven days post-S100B injection, these large, highly stained cells had increased in number throughout the retina. Injection of Aβ and IL-1β also caused an increase in S100B production within the retinal Müller glial cells. Conclusion: These results support the hypothesis that S100B (a glial-derived neurotrophic factor) and IL-1β (a pro-inflammatory cytokine) can modulate the expression and processing of APP in vivo and so may contribute to the progression of Alzheimer’s disease.

Synthesis and biological activities of 2-((heteroaryl)methyl)imidazolines

A series of 2-[(heteroaryl)methyl]imidazolines was synthesized and tested for their activities at α(1)- and α(2)-adrenoceptors and imidazoline I(1) and I(2) receptors. The most active 2-[(indazol-1-yl)methyl]imidazolines showed high or moderate affinities for α(1)- and α(2)-adrenoceptors. However, their intrinsic activities at α(2A)-adrenoceptors proved to be negligible. A selected 7-chloro derivative behaved as a potent α(1)-adrenoceptor antagonist and exhibited peripherally mediated hypotensive effects in rats.

Protective Effects of Non-Anticoagulant Activated Protein C Variant (D36A/L38D/A39V) in a Murine Model of Ischaemic Stroke

Ischaemic stroke is caused by occlusive thrombi in the cerebral vasculature. Although tissue-plasminogen activator (tPA) can be administered as thrombolytic therapy, it has major limitations, which include disruption of the blood-brain barrier and an increased risk of bleeding. Treatments that prevent or limit such deleterious effects could be of major clinical importance. Activated protein C (APC) is a natural anticoagulant that regulates thrombin generation, but also confers endothelial cytoprotective effects and improved endothelial barrier function mediated through its cell signalling properties. In murine models of stroke, although APC can limit the deleterious effects of tPA due to its cell signalling function, its anticoagulant actions can further elevate the risk of bleeding. Thus, APC variants such as APC(5A), APC(Ca-ins) and APC(36-39) with reduced anticoagulant, but normal signalling function may have therapeutic benefit. Human and murine protein C (5A), (Ca-ins) and (36-39) variants were expressed and characterised. All protein C variants were secreted normally, but 5-20% of the protein C (Ca-ins) variants were secreted as disulphide-linked dimers. Thrombin generation assays suggested reductions in anticoagulant function of 50- to 57-fold for APC(36-39), 22- to 27-fold for APC(Ca-ins) and 14- to 17-fold for APC(5A). Interestingly, whereas human wt APC, APC(36-39) and APC(Ca-ins) were inhibited similarly by protein C inhibitor (t½ - 33 to 39 mins), APC(5A) was inactivated ~9-fold faster (t½ - 4 mins). Using the murine middle cerebral artery occlusion ischaemia/repurfusion injury model, in combination with tPA, APC(36-39), which cannot be enhanced by its cofactor protein S, significantly improved neurological scores, reduced cerebral infarct area by ~50% and reduced oedema ratio. APC(36-39) also significantly reduced bleeding in the brain induced by administration of tPA, whereas wt APC did not. If our data can be extrapolated to clinical settings, then APC(36-39) could represent a feasible adjunctive therapy for ischaemic stroke.

Transfer of SAR information from hypotensive indazole to indole derivatives acting at α-adrenergic receptors: In vitro and in vivo studies.

In a search for novel antihypertensive drugs we applied scaffold hopping from the previously described α1-adrenergic receptor antagonists, 1-[(imidazolin-2-yl)methyl]indazoles. The aim was to investigate whether the α-adrenergic properties of the indazole core were transferable to the indole core. The newly obtained 1-[(imidazolin-2-yl)methyl]indole analogues were screened in vitro for their binding affinities for α1-and α2-adrenoceptors, which allowed the identification of the target-based SAR transfer (T_SAR transfer) as well as structure-based SAR transfer (S_SAR transfer) events. However, when screened in vivo with use of anaesthetized male Wistar rats, the new indole ligands showed a different hemodynamic profile than expected. Instead of the immediate hypotensive effect characteristic of peripheral vasodilatator α1 blockers, a biphasic effect was observed, reminiscent of clonidine-like centrally acting antihypertensive agents. This was supported by subsequent in vitro functional studies in [(35)S]GTPγS binding assay, where the indole analogues displayed partial agonist properties at α2-adrenergic receptors. Since no correlation was found between the in vitro binding to α-adrenoceptors and the in vivo hemodynamic effects of the two series of indazole and indole bioisosteric compounds, in a search for new imidazoline-containing adrenergic drugs, the structure-based SAR transfer information obtained from in vitro binding studies should be treated with caution.