Rouhollah Habibey

The Protective Effect of Remote Renal Preconditioning Against Hippocampal Ischemia Reperfusion Injury: Role of KATP Channels

Remote ischemic preconditioning (RIPC), which consists of several brief ischemia/reperfusion applied at the remote site of lethal ischemia reperfusion, can, through activating different mechanisms, increase the ability of the body’s endogenous protection against prolonged ischemia/reperfusion. Recent studies have shown that RIPC has neuroprotective effects, but its mechanisms are not well elucidated. The present study aimed to determine whether activation of KATP channels in remote renal preconditioning decreases hippocampus damage induced by global cerebral ischemia. RIPC was induced by ischemia of the left renal artery (IPC); 24xa0h later, global cerebral ischemia reperfusion (IR) was induced by common carotid arteries occlusion. 5hydroxydecanoate (5HD) and glibenclamide (Gli) were injected before of IPC. The levels of malondialdehyde (MDA) and catalase (CAT) activity were assessed in hippocampus. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) was assessed to detect apoptotic cells in hippocampus. RIPC inhibited apoptosis by decreasing positive TUNEL cells (Pu2009<u20090.05). KATP channels blocking with 5HD and Gli markedly increased apoptosis in hippocampal cells in RIPC group (Pu2009<u20090.001). RIPC decreased MDA level and increased CAT activity in ischemic hippocampus (Pu2009<u20090.01). Also, 5HD and Gli inhibited the effect of RIPC on MDA level and CAT activity (Pu2009<u20090.05). The present study shows that RIPC can effectively attenuate programmed cell death, increase activity of CAT, and reduce MDA levels. Blocking of KATP channels inhibited the protective effects of RIPC.

Incubator-independent cell-culture perfusion platform for continuous long-term microelectrode array electrophysiology and time-lapse imaging

Most in vitro electrophysiology studies extract information and draw conclusions from representative, temporally limited snapshot experiments. This approach bears the risk of missing decisive moments that may make a difference in our understanding of physiological events. This feasibility study presents a simple benchtop cell-culture perfusion system adapted to commercial microelectrode arrays (MEAs), multichannel electrophysiology equipment and common inverted microscopy stages for simultaneous and uninterrupted extracellular electrophysiology and time-lapse imaging at ambient CO2 levels. The concept relies on a transparent, replica-casted polydimethylsiloxane perfusion cap, gravity- or syringe-pump-driven perfusion and preconditioning of pH-buffered serum-free cell-culture medium to ambient CO2 levels at physiological temperatures. The low-cost microfluidic in vitro enabling platform, which allows us to image cultures immediately after cell plating, is easy to reproduce and is adaptable to the geometries of different cell-culture containers. It permits the continuous and simultaneous multimodal long-term acquisition or manipulation of optical and electrophysiological parameter sets, thereby considerably widening the range of experimental possibilities. Two exemplary proof-of-concept long-term MEA studies on hippocampal networks illustrate system performance. Continuous extracellular recordings over a period of up to 70 days revealed details on both sudden and gradual neural activity changes in maturing cell ensembles with large intra-day fluctuations. Correlated time-lapse imaging unveiled rather static macroscopic network architectures with previously unreported local morphological oscillations on the timescale of minutes.

Effects of diphencyprone on expression of Bcl-2 protein in patients with Alopecia areata

Background: Alopecia areata (AA) development is attributed to a T cell involved autoimmune process. Apoptosis is one of the suspected culprits in pathogenesis of this disorder. This disorder can be treated by contact sensitizers like diphencyprone (DPCP). We investigated the effects of treatment with DPCP on the expression of Bcl-2 protein in hair follicle epithelial cells of AA patients and its relation to clinical response to treatment. Materials and Methods: Patients with chronic and extensive AA who had not received any treatment for at least 6 months were included. Furthermore, 3-mm punch biopsies were obtained from the affected areas before starting the treatment, and, six months after DPCP application, punch biopsies of the same size were taken from the following groups of patients: Group 1: six patients with complete hair regrowth, Group 2: six patients with partial regrowth, and Group 3: six patients with no regrowth. The samples were studied by immunohistochemistry to detect and compare the rate of Bcl-2 expression. Results: Level of Bcl-2 expression in respondent patients (Group 1) was significantly higher after DPCP treatment (36.50u2009±u20094.23) compared to pretreatment state (3.67u2009±u20091.406, Pu2009<u20090.001). Similar finding was observed in second group with partial regrowth (17.67u2009±u20091.745 versus 5.33u2009±u20092.076, Pu2009<u20090.01). Such significant change was not observed in third group (4.75u2009±u20091.315 versus 3.50u2009±u20090.645, Pu2009>u20090.05). Conclusion:The results of this study indicate the positive effect of DPCP on regulation (inhibition) of apoptotic process in patients with AA.

THE EFFECT OF ENALAPRIL ON SKIN FLAP VIABILITY IS INDEPENDENT OF ANGIOTENSIN II AT1 RECEPTORS

Random pattern skin flaps are still widely used in plastic surgery. However, necrosis in the distal portion resulting from ischemia is a serious problem, increasing the cost of treatment and hospitalization. To enhance skin flap viability, a variety of pharmacologic agents have been intensively investigated. The aim of this study was to assess the effect of enalapril (an angiotensin-converting enzyme inhibitor) and losartan (an angiotensin receptor blocker) in skin flap viability. Male rats of 200 to 250 g were used. Different doses of enalapril (5, 20, and 50 mg/kg) and losartan (5 mg/kg) were administrated 30 minutes prior to elevate the flap. Flap survival area was evaluated on the seventh postoperative day. Enalapril improved survival area in a dose-dependent manner, but losartan failed to improve survival area, which suggested that the effect of enalapril was not mediated through AT1 receptors.

Antioxidant nanomaterials in advanced diagnoses and treatments of ischemia reperfusion injuries

Organ ischemia with inadequate oxygen supply followed by reperfusion (which initiates a complex of inflammatory responses and oxidative stress) occurs in different clinical conditions and surgical procedures including stroke, myocardial infarction, limb ischemia, renal failure, organ transplantation, free-tissue-transfer, cardiopulmonary bypass, and vascular surgery. Even though pharmacological treatments protect against experimental ischemia reperfusion (I/R) injury, there has not been enough success in their application for patient benefits. The main hurdles in the treatment of I/R injury are the lack of diagnosis tools for understanding the complicated chains of I/R-induced signaling events, especially in the acute phase after ischemia, determining the affected regions of the tissue over time, and then, targeting and safe delivery of antioxidants, drugs, peptides, genes and cells to the areas requiring treatment. Besides the innate antioxidant and free radical scavenging properties, some nanoparticles also show higher flexibility in drug delivery and imaging. This review highlights three main approaches in nanoparticle-mediated targeting of I/R injury: nanoparticles (1) as antioxidants for reducing tissue oxidative stress, (2) for targeted delivery of therapeutic agents to the ischemic regions or cells, and (3) for imaging I/R injury at the molecular, cellular or tissue level and monitoring its evolution using contrasts induced by nanoparticles. These approaches can also be combined to realize so called theranostics for providing simultaneous diagnosis of ischemic regions and treatments by targeted delivery.

A multielectrode array microchannel platform reveals both transient and slow changes in axonal conduction velocity

Due to their small dimensions, electrophysiology on thin and intricate axonal branches in support of understanding their role in normal and diseased brain function poses experimental challenges. To reduce experimental complexity, we coupled microelectrode arrays (MEAs) to bi-level microchannel devices for the long-term in vitro tracking of axonal morphology and activity with high spatiotemporal resolution. Our model allowed the long-term multisite recording from pure axonal branches in a microscopy-compatible environment. Compartmentalizing the network structure into interconnected subpopulations simplified access to the locations of interest. Electrophysiological data over 95 days in vitro (DIV) showed an age-dependent increase of axonal conduction velocity, which was positively correlated with, but independent of evolving burst activity over time. Conduction velocity remained constant at chemically increased network activity levels. In contrast, low frequency (1u2009Hz, 180 repetitions) electrical stimulation of axons or network subpopulations evoked amplitude-dependent direct (5–35 ms peri-stimulus) and polysynaptic (35–1,000 ms peri-stimulus) activity with temporarily (<35 ms) elevated propagation velocities along the perisomatic branches. Furthermore, effective stimulation amplitudes were found to be significantly lower (>250u2009mV) in microchannels when compared with those reported for unconfined cultures (>800u2009mV). The experimental paradigm may lead to new insights into stimulation-induced axonal plasticity.

Natural lecithin promotes neural network complexity and activity

Phospholipids in the brain cell membranes contain different polyunsaturated fatty acids (PUFAs), which are critical to nervous system function and structure. In particular, brain function critically depends on the uptake of the so-called “essential” fatty acids such as omega-3 (n-3) and omega-6 (n-6) PUFAs that cannot be readily synthesized by the human body. We extracted natural lecithin rich in various PUFAs from a marine source and transformed it into nanoliposomes. These nanoliposomes increased neurite outgrowth, network complexity and neural activity of cortical rat neurons in vitro. We also observed an upregulation of synapsin I (SYN1), which supports the positive role of lecithin in synaptogenesis, synaptic development and maturation. These findings suggest that lecithin nanoliposomes enhance neuronal development, which may have an impact on devising new lecithin delivery strategies for therapeutic applications.

Preconditioning with morphine protects hippocampal CA1 neurons from ischemia–reperfusion injury via activation of the mTOR pathway

The signaling pathway of chronic morphine treatment to prevent neuronal damage following transient cerebral ischemia is not clear. In this study, we examined the role of mammalian target of rapamycin (mTOR) to identify the neuroprotective effects of chronic morphine preconditioning on the hippocampus following ischemia-reperfusion (I/R) injury. Morphine was administered for 5 days, twice a day, before inducing I/R injury. The possible role of mTOR was evaluated by the injection of rapamycin (5 mg/kg body weight, by intraperitoneal injection) before I/R was induced. The passive avoidance test was used to evaluate memory performance. Neuronal density and apoptosis were measured in the CA1 region, 72 h after I/R injury. The expressions of mTOR and phosphorylated mTOR (p-mTOR), as well as superoxide dismutase (SOD) activity were determined 24 h after I/R injury. Chronic morphine treatment attenuated apoptosis and neuronal loss in the hippocampus after I/R injury, which led to improvement in memory (P < 0.05 vs. untreated I/R) and increase in the expression of p-mTOR (P < 0.05 vs. untreated I/R) and SOD activity (P < 0.05 vs. untreated I/R) in the hippocampus. Pretreatment with rapamycin abolished all the above-mentioned protective effects. These results describe novel findings whereby chronic morphine preconditioning in hippocampal CA1 neurons is mediated by the mTOR pathway, and through increased phosphorylation of mTOR can alleviate oxidative stress and apoptosis, and eventually protect the hippocampus from I/R injury.

Activation of mitochondrial KATP channels mediates neuroprotection induced by chronic morphine preconditioning in hippocampal CA-1 neurons following cerebral ischemia

PURPOSEnPharmacologic preconditioning, through activating several mechanisms and mediators, can increase the tolerance of different tissues against ischemia/reperfusion (I/R) injury. Recent studies have shown that morphine preconditioning has protective effects in different organs, especially in the heart. Nevertheless, its mechanisms are not well elucidated in the brain. The present study aimed to clarify whether the activation of mitochondrial KATP (mKATP) channels in chronic morphine (CM) preconditioning could decrease hippocampus damage following I/R injury.nnnMATERIALS AND METHODSnCM preconditioning was performed by the administration of additive doses of morphine for 5days before I/R injury induction. I/R injury was induced by the occlusion of bilateral common carotid arteries. The possible role of mKATP channels was evaluated by the injection of 5-hydroxydecanoate (5-HD) before I/R injury. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) was performed to detect apoptosis in hippocampal neurons. The expressions of B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (BAX) and levels of malondialdehyde (MDA) and catalase (CAT) enzymes were assessed.nnnRESULTSnCM attenuated apoptosis in the hippocampal CA1 neurons (P<0.001 vs I/R), and mKATP channel blocking with 5-HD significantly increased apoptosis (P<0.001 vs CM+I/R). CM increased CAT activity (P<0.05 vs I/R) and Bcl-2 protein expression (P<0.01 vs I/R), while it decreased MDA level (P<0.05 vs I/R) and BAX protein expression (P<0.05 vs I/R). Pretreatment with 5-HD abolished all the above-mentioned effects of CM.nnnCONCLUSIONSnThese findings describe novel evidence whereby CM preconditioning in hippocampal CA1 neurons can improve oxidative stress and apoptosis through the activation of mKATP channels and eventually protect the hippocampal tissue against I/R injury.

Microchannel Scaffolds for Neural Signal Acquisition and Analysis

Replica-casting finds wide application in soft lithography and microfluidics. Most commonly, structures are molded with micro- to nano-patterned photoresists as master casts into polydimethylsiloxane (PDMS). PDMS features many favorable properties. It reproduces geometric details with nanometer fidelity, has low cytotoxicity and is transparent in the visible spectrum. It is furthermore biostable both in vitro and in vivo, can be plasma-bonded to itself, has low water permeability and is easy to handle and process. After curing, the PDMS can be peeled from the master and latter usually be reused if patterns are not undercut. Here, we describe the straightforward replica-molding process for devices that can be exploited either as perforated microchannel scaffolds for the in vitro use in axonal guidance and regeneration studies on microelectrode arrays (MEAs) or for the production of tissue-conformal in vivo MEAs for neuroprosthetic applications.