Microfluidics for Neuronal Cell and Circuit Engineering.

The widespread adoption of microfluidic devices among the neuroscience and neurobiology communities has enabled addressing a broad range of questions at the molecular, cellular, circuit, and system levels. Here, we review biomedical engineering approaches that harness the power of microfluidics for bottom-up generation of neuronal cell types and for the assembly and analysis of neural circuits. Microfluidics-based approaches are instrumental to generate the knowledge necessary for the derivation of diverse neuronal cell types from human pluripotent stem cells, as they enable the isolation and subsequent examination of individual neurons of interest. Moreover, microfluidic devices allow to engineer neural circuits with specific orientations and directionality by providing control over neuronal cell polarity and permitting the isolation of axons in individual microchannels. Similarly, the use of microfluidic chips enables the construction not only of 2D but also of 3D brain, retinal, and peripheral nervous system model circuits. Such brain-on-a-chip and organoid-on-a-chip technologies are promising platforms for studying these organs as they closely recapitulate some aspects of in vivo biological processes. Microfluidic 3D neuronal models, together with 2D in vitro systems, are widely used in many applications ranging from drug development and toxicology studies to neurological disease modeling and personalized medicine. Altogether, microfluidics provide researchers with powerful systems that complement and partially replace animal models.

Neuronal Network Topology Indicates Distinct Recovery Processes after Stroke.

Despite substantial recent progress in network neuroscience, the impact of stroke on the distinct features of reorganizing neuronal networks during recovery has not been defined. Using a functional connections-based approach through 2-photon in vivo calcium imaging at the level of single neurons, we demonstrate for the first time the functional connectivity maps during motion and nonmotion states, connection length distribution in functional connectome maps and a pattern of high clustering in motor and premotor cortical networks that is disturbed in stroke and reconstitutes partially in recovery. Stroke disrupts the network topology of connected inhibitory and excitatory neurons with distinct patterns in these 2 cell types and in different cortical areas. These data indicate that premotor cortex displays a distinguished neuron-specific recovery profile after stroke.

Optogenetics for neural transplant manipulation and functional analysis.

Transplantation of neural stem cells (NSCs) or NSC-derived neurons into the brain is a promising therapeutic approach to restore neuronal function. Rapid progress in the NSCs research field, particularly due to the exploitation of induced pluripotent stem cells (iPSCs), offers great potential and an unlimited source of stem cell-derived neural grafts. Studying the functional integration of these grafts into host brain tissues and their effects on each other have been boosted by the implementation of optogenetic technologies. Optogenetics provides high spatiotemporal functional manipulations of grafted or host neurons in parallel. This review aims to highlight the impact of optogenetics in neural stem cell transplantations.

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.

The Positive Role of Curcumin-Loaded Salmon Nanoliposomes on the Culture of Primary Cortical Neurons.

Curcumin (diferuloylmethane) is a natural bioactive compound with many health-promoting benefits. However, its poor water solubility and bioavailability has limited curcumin’s biomedical application. In the present study, we encapsulated curcumin into liposomes, formed from natural sources (salmon lecithin), and characterized its encapsulation efficiency and release profile. The proposed natural carriers increased the solubility and the bioavailability of curcumin. In addition, various physico-chemical properties of the developed soft nanocarriers with and without curcumin were studied. Nanoliposome-encapsulated curcumin increased the viability and network formation in the culture of primary cortical neurons and decreased the rate of apoptosis.

Engineered modular neuronal networks-on-chip represent structure-function relationship.

Brain function is substantially linked to the highly organized modular structure of neuronal networks. However, the structure of in vitro assembled neuronal circuits often exhibits variability, complicating the consistent recording of network functional output and its correlation to network structure. Therefore, engineering neuronal structures with predefined geometry and reproducible functional features is essential to precisely model in vivo neuronal circuits. Here, we engineered microchannel devices to assemble 2D and 3D modular networks. The microchannel devices were coupled with a multi-electrode array (MEA) electrophysiology system to enable recordings from circuits. Each network consisted of 64 modules connected to their adjacent modules by micron-sized channels. Modular circuits within microchannel devices showed enhanced activity and functional connectivity traits. This includes metrics such as connection weights, clustering coefficient, global efficiency, and the number of hub neurons with higher betweenness centrality. In addition, modular networks demonstrated an increased functional modularity score compared to the randomly formed circuits. Neurons within individual modules displayed uniform network characteristics and predominantly participated in their respective functional communities within the same or neighboring physical modules. These observations highlight that the modular network structure promotes the development of segregated functional connectivity traits while simultaneously enhancing the efficiency of overall network connectivity. Our findings emphasize the significant impact of physical constraints on the activity patterns and functional organization within engineered modular networks. These circuits, characterized by stable modular architecture and intricate functional dynamics-key features of the brain networks-offer a robust in vitro model for advancing neuroscience research.

Incubator-independent perfusion system integrated with microfluidic device for continuous electrophysiology and microscopy readouts.

Advances in primary and stem cell derived neuronal cell culture techniques and abundance of available neuronal cell types have enabledin vitroneuroscience as a substantial approach to modelin vivoneuronal networks. Survival of the cultured neurons is inevitably dependent on the cell culture incubators to provide stable temperature and humidity and to supply required CO2levels for controlling the pH of culture medium. Therefore, imaging and electrophysiology recordings outside of the incubator are often limited to the short-term experimental sessions. This restricts our understanding of physiological events to the short snapshots of recorded data while the major part of temporal data is neglected. Multiple custom-made and commercially available platforms like integrated on-stage incubators have been designed to enable long-term microscopy. Nevertheless, long-term high-spatiotemporal electrophysiology recordings from developing neuronal networks needs to be addressed. In the present work an incubator-independent polydimethylsiloxane-based double-wall perfusion chamber was designed and integrated with multi-electrode arrays (MEAs) electrophysiology and compartmentalized microfluidic device to continuously record from engineered neuronal networks at sub-cellular resolution. Cell culture media underwent iterations of conditioning to the ambient CO2and adjusting its pH to physiological ranges to retain a stable pH for weeks outside of the incubator. Double-wall perfusion chamber and an integrated air bubble trapper reduced media evaporation and osmolality drifts of the conditioned media for two weeks. Aligned microchannel-microfluidic device on MEA electrodes allowed neurite growth on top of the planar electrodes and amplified their extracellular activity. This enabled continuous sub-cellular resolution imaging and electrophysiology recordings from developing networks and their growing neurites. The on-chip versatile and self-contained system provides long-term, continuous and high spatiotemporal access to the network data and offers a robustin vitroplatform with many potentials to be applied on advanced cell culture systems including organ-on-chip and organoid models.

Human neural network activity reacts to gravity changes in vitro.

During spaceflight, humans experience a variety of physiological changes due to deviations from familiar earth conditions. Specifically, the lack of gravity is responsible for many effects observed in returning astronauts. These impairments can include structural as well as functional changes of the brain and a decline in cognitive performance. However, the underlying physiological mechanisms remain elusive. Alterations in neuronal activity play a central role in mental disorders and altered neuronal transmission may also lead to diminished human performance in space. Thus, understanding the influence of altered gravity at the cellular and network level is of high importance. Previous electrophysiological experiments using patch clamp techniques and calcium indicators have shown that neuronal activity is influenced by altered gravity. By using multi-electrode array (MEA) technology, we advanced the electrophysiological investigation covering single-cell to network level responses during exposure to decreased (micro-) or increased (hyper-) gravity conditions. We continuously recorded in real-time the spontaneous activity of human induced pluripotent stem cell (hiPSC)-derived neural networks in vitro. The MEA device was integrated into a custom-built environmental chamber to expose the system with neuronal cultures to up to 6 g of hypergravity on the Short-Arm Human Centrifuge at the DLR Cologne, Germany. The flexibility of the experimental hardware set-up facilitated additional MEA electrophysiology experiments under 4.7 s of high-quality microgravity (10-6 to 10-5 g) in the Bremen drop tower, Germany. Hypergravity led to significant changes in activity. During the microgravity phase, the mean action potential frequency across the neural networks was significantly enhanced, whereas different subgroups of neurons showed distinct behaviors, such as increased or decreased firing activity. Our data clearly demonstrate that gravity as an environmental stimulus triggers changes in neuronal activity. Neuronal networks especially reacted to acute changes in mechanical loading (hypergravity) or de-loading (microgravity). The current study clearly shows the gravity-dependent response of neuronal networks endorsing the importance of further investigations of neuronal activity and its adaptive responses to micro- and hypergravity. Our approach provided the basis for the identification of responsible mechanisms and the development of countermeasures with potential implications on manned space missions.

Skeletal muscle post-conditioning by diazoxide, anti-oxidative and anti-apoptotic mechanisms.

Pretreatment with diazoxide, K(ATP) channel opener, increases tissue tolerance against ischemia reperfusion (IR) injury. In clinical settings pretreatment is rarely an option therefore we evaluated the effect of post-ischemic treatment with diazoxide on skeletal muscle IR injury. Rats were treated with either saline, diazoxide (K(ATP) opener; 40 mg/kg) or 5-hydroxydecanoate (5-HD; mitochondrial K(ATP) inhibitor; 40 mg/kg) after skeletal muscle ischemia (3 h) and reperfusion (6, 24 or 48 h). Tissue contents of malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) activities, Bax and Bcl-2 protein expression and muscle histology were determined. Apoptosis was examined (24 and 48 h) after ischemia. IR induced severe histological damage, increased MDA content and Bax expression (24 and 48 h; p < 0.01) and decreased CAT and SOD activities (6 and 24 h, p < 0.01 and 48 h, p < 0.05), with no significant effect on Bcl-2 expression. Diazoxide reversed IR effects on MDA (6 and 24 h; p < 0.05), SOD (6 and 24 h; p < 0.01) and CAT (6 and 48 h, p < 0.05 and 24 h p < 0.01) and tissue damage. Diazoxide also decreased Bax (24 and 48 h; p < 0.05) and increased Bcl-2 protein expression (24 and 48 h; p < 0.01). Post-ischemic treatment with 5-HD had no significant effect on IR injury. Number of apoptotic nuclei in IR and 5-HD treated groups significantly increased (p < 0.001) while diazoxide decreased apoptosis (p < 0.01). The results suggested that post-ischemic treatment with diazoxide decrease oxidative stress in acute phase which modulates expression of apoptotic proteins in the late phase of reperfusion injury. Involvement of KATP channels in this effect require further evaluations.

Late anti-apoptotic effect of K(ATP) channel opening in skeletal muscle.

Necrosis and apoptosis caused by ischaemia-reperfusion (IR) result in myocyte death and atrophy. ATP-sensitive K(+) (K(ATP) ) channels activation increases tissue tolerance of IR-injury. Thus, in the present study, we evaluated the effects of K(ATP) channel activation on skeletal muscle apoptosis after IR. Male Wistar rats were treated with 40 mg/kg, i.p., diazoxide (a K(ATP) channel opener) or 5 mg/kg, i.p., glibenclamide (a K(ATP) channel inhibitor) 30 min before the induction of 3 h ischaemia, followed by 6, 24 or 48 h reperfusion. At the end of the reperfusion period, the gastrocnemius muscle was removed for the analysis of tissue malondialdehyde content, superoxide dismutase (SOD) and catalase (CAT) activity, Bax and Bcl-2 protein expression, histological damage and the number of apoptotic nuclei. Ischaemia-reperfusion increased malondialdehyde content (P < 0.01) and Bax expression (P < 0.01) and induced severe histological damage, in addition to decreasing CAT and SOD activity (P < 0.01 and P < 0.05, respectively) and Bcl-2 expression (P < 0.01). Diazoxide reversed the effects of IR on tissue damage, MDA content, SOD and CAT activity (after 6 and 24 h reperfusion; P < 0.05) and Bax and Bcl-2 expression (after 24 and 48 h reperfusion; P < 0.01). In contrast, glibenclamide pretreatment had no effect. The number of apoptotic nuclei in the IR and glibenclamide-pretreated groups increased significantly (P < 0.001 vs Sham). In contrast, diazoxide pretreatment decreased the number of apoptotic nuclei compared with the IR group (P < 0.01). The results of the present study suggest that the K(ATP) channel activator diazoxide attenuates lipid peroxidation during the first hour of reperfusion and modulates apoptotic pathways at later time points.

Optogenetic Control of Human Stem Cell-Derived Neurons.

Spontaneous and optogenetically evoked activities of human induced pluripotent stem cell (hiPSC)-derived neurons can be assessed by patch clamp and multi-electrode array (MEA) electrophysiology. Optogenetic activation of these human neurons facilitates the characterization of their functional properties at the single neuron and circuit level. Here we showcase the preparation of hiPSC-derived neurons expressing optogenetic actuators, in vitro optogenetic stimulation and simultaneous functional recordings using patch clamp and MEA electrophysiology.

Long-term morphological and functional dynamics of human stem cell-derived neuronal networks on high-density micro-electrode arrays.

Comprehensive electrophysiological characterizations of human induced pluripotent stem cell (hiPSC)-derived neuronal networks are essential to determine to what extent these in vitro models recapitulate the functional features of in vivo neuronal circuits. High-density micro-electrode arrays (HD-MEAs) offer non-invasive recording with the best spatial and temporal resolution possible to date. For 3 months, we tracked the morphology and activity features of developing networks derived from a transgenic hiPSC line in which neurogenesis is inducible by neurogenic transcription factor overexpression. Our morphological data revealed large-scale structural changes from homogeneously distributed neurons in the first month to the formation of neuronal clusters over time. This led to a constant shift in position of neuronal cells and clusters on HD-MEAs and corresponding changes in spatial distribution of the network activity maps. Network activity appeared as scarce action potentials (APs), evolved as local bursts with longer duration and changed to network-wide synchronized bursts with higher frequencies but shorter duration over time, resembling the emerging burst features found in the developing human brain. Instantaneous firing rate data indicated that the fraction of fast spiking neurons (150-600 Hz) increases sharply after 63 days post induction (dpi). Inhibition of glutamatergic synapses erased burst features from network activity profiles and confirmed the presence of mature excitatory neurotransmission. The application of GABAergic receptor antagonists profoundly changed the bursting profile of the network at 120 dpi. This indicated a GABAergic switch from excitatory to inhibitory neurotransmission during circuit development and maturation. Our results suggested that an emerging GABAergic system at older culture ages is involved in regulating spontaneous network bursts. In conclusion, our data showed that long-term and continuous microscopy and electrophysiology readouts are crucial for a meaningful characterization of morphological and functional maturation in stem cell-derived human networks. Most importantly, assessing the level and duration of functional maturation is key to subject these human neuronal circuits on HD-MEAs for basic and biomedical applications.

Tracking connectivity maps in human stem cell-derived neuronal networks by holographic optogenetics.

Neuronal networks derived from human induced pluripotent stem cells have been exploited widely for modeling neuronal circuits, neurological diseases, and drug screening. As these networks require extended culturing periods to functionally mature in vitro, most studies are based on immature networks. To obtain insights on long-term functional features, we improved a glia-neuron co-culture protocol within multi-electrode arrays, facilitating continuous assessment of electrical features in weekly intervals. By full-field optogenetic stimulation, we detected an earlier onset of neuronal firing and burst activity compared with spontaneous activity. Full-field stimulation enhanced the number of active neurons and their firing rates. Compared with full-field stimulation, which evoked synchronized activity across all neurons, holographic stimulation of individual neurons resulted in local activity. Single-cell holographic stimulation facilitated to trace propagating evoked activities of 400 individually stimulated neurons per multi-electrode array. Thereby, we revealed precise functional neuronal connectivity motifs. Holographic stimulation data over time showed increasing connection numbers and strength with culture age. This holographic stimulation setup has the potential to establish a profound functional testbed for in-depth analysis of human-induced pluripotent stem cell-derived neuronal networks.

Expression of Bcl-2 and Bax after hippocampal ischemia in DHA + EPA treated rats.

To determine the impact of ω3 fatty acids on post-ischemic expression of pro- and anti-apoptotic proteins in hippocampus, male rats were received 10 or 100 mg/kg [Docosahexaenoic acid (DHA) + Ecosapentaenoic acid (EPA); gavage; 21 days before ischemia to 2-10 days after ischemia]. Global cerebral ischemia reperfusion (IR) was performed using the four-vessel occlusion model; ischemia 8 min and reperfusion 6, 48 h and 10 days. IR increased Bcl-2 and Bax expression after 48 h (p < 0.05 and p < 0.01 vs. sham) and 10 days (only Bax; p < 0.05), without significant difference with DHA + EPA groups after 6 h. But after 48 h expression of Bcl-2 increased (p < 0.05 vs. IR) and Bax decreased (p < 0.05). At day 10 after ischemia expression of Bax in DHA + EPA acid groups was less than IR (p < 0.05) and in 100 mg/kg DHA + EPA group Bcl-2 expression was more than IR (p < 0.05). These data suggested that long-term gavage with DHA + EPA increase hippocampal neurons survival for days after ischemia, revealed by increased Bcl-2 and decreased Bax expressions.

Effect of crocus sativus on gentamicin induced nephrotoxicity.

Crocus sativus, known as saffron, is used in folk medicine for treatment of different types of diseases, and its anti-inflammatory and free radical scavenging activities have been demonstrated. The present study evaluated gentamicin nephrotoxicity in saffron treated rats. Male Wistar rats (200-250 g) were treated with saffron (40 or 80 mg/k/d) for 10 days, or saffron (40 or 80 mg/ kg/d) for 10 days and gentamicin 80 mg/kg/d for five days, starting from day 6. At the end of treatment, blood samples were taken for measurement of serum creatinine (SCr) and BUN. The left kidney was prepared for histological evaluation and the right kidney for Malondialdehyde (MDA) measurement. Gentamicin 80 (mg/k/d) increased SCr, BUN and renal tissue levels of MDA and induced severe histological changes. Saffron at 40 mg/k/d significantly reduced gentamicin-induced increases in BUN and histological scores (p<0.05). Gentamicin-induced increases in BUN, SCr and MDA and histological injury were significantly reduced by treatment with saffron 80 mg/k/d (p<0.05, p<0.001, p<0.05, and p<0.001 respectively). In conclusion, our results suggest that saffron treatment reduces gentamicin-induced nephrotoxicity and this effect seems to be dose dependent.

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 (36.50 +/- 4.23) compared to pretreatment state (3.67 +/- 1.406, P < 0.001). Similar finding was observed in second group with partial regrowth (17.67 +/- 1.745 versus 5.33 +/- 2.076, P < 0.01). Such significant change was not observed in third group (4.75 +/- 1.315 versus 3.50 +/- 0.645, P > 0.05). 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.

Interaction of mTOR and iNOS pathways in protection against Ischemia/Reperfusion injury.

Chronic morphine (CM) treatment increases the phosphorylation of the mammalian target of rapamycin (mTOR), which confers neuroprotection against ischemia/reperfusion (I/R) injury. Besides its important regulatory role in the proliferation, metabolism, and survival of cells, the mTOR is critically involved in intracellular signaling events during I/R injury. In the present study, we investigated the interaction between the expressions of the mTOR and inducible nitric oxide synthase (iNOS) and their possible protective effects on hippocampal neurons against I/R injury in morphine-dependent mice. Additive doses of morphine were administered for 5 days to BALB/c mice so as to induce CM preconditioning before I/R injury. Global brain ischemia was induced via the occlusion of bilateral common carotid arteries for 30 min. CM attenuated iNOS expression, NO production, and malondialdehyde activity in the hippocampal tissue. Pretreatment with rapamycin, the inhibitor of mTOR, abolished all the above mentioned effects of CM. These findings suggested that CM acted through the mTOR signaling pathways to regulate iNOS expression and oxidative state in the hippocampal tissue after I/R injury.

Selenium nanoparticles for targeted stroke therapy through modulation of inflammatory and metabolic signaling.

Ischemic cerebral stroke is a major cause of death and morbidity. Currently, no neuroprotective agents have been shown to impact the clinical outcomes in cerebral stroke cases. Here, we report therapeutic effects of Se nanoparticles on ischemic stroke in a murine model. Anti-transferrin receptor monoclonal antibody (OX26)-PEGylated Se nanoparticles (OX26-PEG-Se NPs) were designed and synthesized and their neuroprotective effects were measured using in vitro and in vivo approaches. We demonstrate that administration of the biodegradable nanoparticles leads to resolution of brain edema, protection of axons in hippocampus region, and myelination of hippocampal area after cerebral ischemic stroke. Our nanoparticle design ensures efficient targeting and minimal side effects. Hematological and biochemical analyses revealed no undesired NP-induced changes. To gain mechanistic insights into the therapeutic effects of these particles, we characterized the changes to the relevant inflammatory and metabolic signaling pathways. We assessed metabolic regulator mTOR and related signaling pathways such as hippo, Ubiquitin-proteasome system (ERK5), Tsc1/Tsc2 complex, FoxO1, wnt/ß-catenine signaling pathway. Moreover, we examined the activity of jak2/stat3 signaling pathways and Adamts1, which are critically involved in inflammation. Together, our study provides a promising treatment strategy for cerebral stroke based on Se NP induced suppression of excessive inflammation and oxidative metabolism.

Morphine dependence protects rat kidney against ischaemia-reperfusion injury.

Ischaemic preconditioning (IPC) protects the heart and kidneys against ischaemia-reperfusion (I/R) injury. It has been shown that opioid receptor activation can mimic cardiac IPC. In a kidney model of I/R, a single dose of morphine failed to mimic IPC. The aim of the present study was to determine the role of chronic morphine (dependence) in protection against renal I/R injury. Male Wistar rats were treated with increasing doses of morphine (20-30 mg/kg per day, s.c., for 5 days) to develop morphine dependence (MD). Three weeks before the I/R procedure, the right kidney was removed. Ischaemia-reperfusion injury was induced by clamping the left renal artery for 45 min, followed by 24 h reperfusion. Some MD rats were pretreated with naloxone (5 mg/kg, s.c.). Twenty-four hours later, creatinine and sodium concentrations were measured in serum and urine, then creatinine clearance (CCr) and the fractional excretion of sodium (FE(Na)) were calculated. Blood urea nitrogen (BUN) was measured only in serum samples. Kidneys were also assessed histologically for evidence of tissue injury. In the present study, MD decreased tissue injury (histological score), serum creatinine and BUN levels, increased CCr and decreased FE(Na) after I/R. Pretreatment with naloxone attenuated the protective effects of MD. Morphine dependence did not have any significant effect on urine volume. In conclusion, it seems that morphine dependence protects the kidney against I/R injury via opioid receptor-dependent pathways. Further studies are required to clearly determine the mechanisms involved.