Ljiljana Krizanac-Bengez

A new dynamic in vitro model for the multidimensional study of astrocyte-endothelial cell interactions at the blood-brain barrier.

Blood-brain barrier endothelial cells are characterized by the presence of tight intercellular junctions, the absence of fenestrations, and a paucity of pinocytotic vesicles. The in vitro study of the BBB has progressed rapidly over the past several years as new cell culture techniques and improved technologies to monitor BBB function became available. Studies carried out on viable in vitro models are set to accelerate the design of drugs that selectively and aggressively can target the CNS. Several systems in vitro attempt to reproduce the physical and biochemical behavior of intact BBB, but most fail to reproduce the three-dimensional nature of the in vivo barrier and do not allow concomitant exposure of endothelial cells to abluminal (glia) and lumenal (flow) influences. For this purpose, we have developed a new dynamic in vitro BBB model (NDIV-BBB) designed to allow for extensive pharmacological, morphological and physiological studies. Bovine aortic endothelial cells (BAEC) developed robust growth and differentiation when co-cultured alone. In the presence of glial cells, BAEC developed elevated Trans-Endothelial Electrical Resistance (TEER). Excision of individual capillaries proportionally decreased TEER; the remaining bundles were populated with healthy cells. Flow played an essential role in EC differentiation by decreasing cell division. In conclusion, this new dynamic model of the BBB allows for longitudinal studies of the effects of flow and co-culture in a controlled and fully recyclable environment that also permits visual inspection of the abluminal compartment and manipulation of individual capillaries.

Mechanisms of glucose transport at the blood-brain barrier: an in vitro study.

How the brain meets its continuous high metabolic demand in light of varying plasma glucose levels and a functional blood-brain barrier (BBB) is poorly understood. GLUT-1, found in high density at the BBB appears to maintain the continuous shuttling of glucose across the blood-brain barrier irrespective of the plasma concentration. We examined the process of glucose transport across a quasi-physiological in vitro blood-brain barrier model. Radiolabeled tracer permeability studies revealed a concentration ratio of abluminal to luminal glucose in this blood-brain barrier model of approximately 0.85. Under conditions where [glucose](lumen) was higher than [glucose](ablumen), influx of radiolabeled 2-deoxyglucose from lumen to the abluminal compartment was approximately 35% higher than efflux from the abluminal side to the lumen. However, when compartmental [glucose] were maintained equal, a reversal of this trend was seen (approximately 19% higher efflux towards the lumen), favoring establishment of a luminal to abluminal concentration gradient. Immunocytochemical experiments revealed that in addition to segregation of GLUT-1 (luminal>abluminal), the intracellular enzyme hexokinase was also asymmetrically distributed (abluminal>luminal). We conclude that glucose transport at the CNS/blood interface appears to be dependent on and regulated by a serial chain of membrane-bound and intracellular transporters and enzymes.

Effects of transient loss of shear stress on blood-brain barrier endothelium : Role of nitric oxide and IL-6

Loss of blood-brain barrier (BBB) function may contribute to post-ischemic cerebral injury by yet unknown mechanisms. Ischemia is associated with anoxia, aglycemia and loss of flow (i.e. shearing forces). We tested the hypothesis that loss of shear stress alone does not acutely affect BBB function due to a protective cascade of mechanisms involving cytokines and nitric oxide (NO). To determine the relative contribution of shear stress on BBB integrity we used a dynamic in vitro BBB model based on co-culture of rat brain microvascular endothelial cells (RBMEC) and astrocytes. Trans-endothelial electrical resistance (TEER), IL-6 release and NO levels were measured from the lumenal and ablumenal compartments throughout the experiment. Flow-exposed RBMEC were challenged with 1 h of normoxic-normoglycemic flow cessation (NNFC) followed by reperfusion for 2 to 24 h. NNFC caused a progressive drop in nitric oxide production during flow cessation followed by a time-dependent increase in ablumenal IL-6 associated with a prolonged NO increase during reperfusion. The nitric oxide synthetase (NOS) inhibitor L-NAME (10 microM) abrogated all effects of NNFC, including changes in NO and cytokine production. BBB permeability did not increase during or after NNFC/reperfusion, but was increased by treatment with L-NAME or when the effects of IL-6 were blocked. Flow adapted RBMEC and astrocytes respond to NNFC/reperfusion by overproduction of IL-6, possibly secondary to increased production of NO during the reperfusion. Maintenance of BBB function during and following NNFC appears to depend on intact NO signaling and IL-6 release.

Loss of shear stress induces leukocyte-mediated cytokine release and blood-brain barrier failure in dynamic in vitro blood-brain barrier model.

Brain ischemia is associated with an acute release of pro‐inflammatory cytokines, notably TNF‐α and IL‐6 and failure of the blood–brain barrier. Shear stress, hypoxia‐hypoglycemia, and blood leukocytes play a significant role in blood–brain barrier failure during transient or permanent ischemia. However, these mechanisms have not been studied as independent variables for in vitro ischemia. The present study, using a dynamic in vitro blood–brain barrier model, showed that flow cessation/reperfusion under normoxia–normoglycemia or hypoxia–hypoglycemia without blood leukocytes in the luminal perfusate had a modest, transient effect on cytokine release and blood–brain barrier permeability. By contrast, exposure to normoxic–normoglycemic flow cessation/reperfusion with blood leukocytes in the luminal perfusate led to a significant increase in TNF‐α and IL‐6, accompanied by biphasic blood–brain barrier opening. Enhanced permeability was partially prevented with an anti‐TNF‐α antibody. In leukocyte‐free cartridges, the same levels of IL‐6 had no effect, while TNF‐α caused a moderate increase in blood–brain barrier permeability, suggesting that blood leukocytes are the prerequisite for cytokine release and blood–brain barrier failure during reduction or cessation of flow. These cells induce release of TNF‐α early after ischemia/reperfusion; TNF‐α triggers release of IL‐6, since blockade of TNF‐α prevents IL‐6 release, whereas blockade of IL‐6 induces TNF‐α release. Pre‐treatment of blood leukocytes with the cyclooxygenase (COX) inhibitor, ibuprofen, inhibited cytokine release and completely preserved blood–brain barrier permeability during the reperfusion period. In conclusion, loss of flow (flow cessation/reperfusion) independent of hypoxia–hypoglycemia plays a significant role in blood–brain barrier failure by stimulating leukocyte‐mediated inflammatory mechanisms.

Mechanisms of endothelial survival under shear stress.

Endothelial cells (ECs) are exposed to cytotoxic reactive oxygen species and oxidation products of NO, yet they are characterized by low apoptotic rates and have an average life span of many years. EC exposure to flow has been shown to downregulate cell cycle-related genes and cause cytoskeletal rearrangement. We hypothesized that exposure to flow also causes molecular and physiological changes that induce antioxidant properties in ECs. We used cDNA array expression profiling and protein analysis to study the responses of human ECs exposed to flow in a hollow fiber apparatus or the same ECs grown under static conditions. Our results show that shear-induced synchronized expression of processes control oxidant production; these changes included upregulation of NADH-producing enzymes (Krebs cycle dehydrogenases and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) accompanied by simultaneous decrease in NADH-depleting pathways (e.g., lactate dehydrogenase [LDH]) and diminished production of lactate. Exposure to flow upregulated cytoskeletal genes. Our results suggest that, in addition to inhibition of cell cycle, exposure to flow influences ECs by controlling expression of enzymes involved in the generation of antioxidant intermediates and in adaptive control of cell shape. These changes may explain longevity and antioxidant efficiency of ECs and may provide insight in mechanisms leading to pathological conditions such as arteriosclerosis.

A Randomized, Placebo-Controlled Trial of Transdiscal Radiofrequency, Biacuplasty for Treatment of Discogenic Lower Back Pain

OBJECTIVE The aim was to compare the efficacy of intradiscal biacuplasty (IDB) with that of placebo treatment for discogenic low back pain. DESIGN This is a randomized, placebo-controlled trial. Subjects were randomized on a 1:1 basis to IDB and sham groups. Follow-ups were conducted at 1, 3, and 6 months. Subjects and coordinators were blinded to randomization until 6 months. Of the 1,894 subjects screened, 64 subjects were enrolled, and 59 were treated: 29 randomized to IDB and 30 to sham. All subjects had a history of chronic low back pain for longer than 6 months. INTERVENTIONS Two cooled radiofrequency (RF) electrodes placed in a bipolar manner in affected discs to lesion the nociceptive fibers of the annulus fibrosus. The sham procedure was identical to the active treatment except that probes were not directly inserted into the disc space, and RF energy was not actively delivered. RESULTS The principal outcome measures were physical function, pain, disability, and opioid usage. Patients in the IDB group exhibited statistically significant improvements in physical function (P = 0.029), pain (P = 0.006), and disability (P = 0.037) at 6-month follow-up as compared to patients who received sham treatment. Treatment patients reported a reduction of 16 mg daily intake of opioids at 6 months; however, the results were not statistically different from sham patients. CONCLUSIONS The results suggest that the clinical benefits observed in this study are the result of non-placebo treatment effects afforded by IDB. IDB should be recommended to select the patients with chronic discogenic low back pain. (Clinicaltrials.gov number, NCT00750191.).

Use of a three-dimensional in vitro model of the rat blood-brain barrier to assay nucleoside efflux from brain.

Extracellular adenosine is produced in brain during physiological and pathophysiological conditions. Once produced, this adenosine can undergo one or more of the following fates: it can interact with its receptors, it can be scavenged by astrocytes and/or neurons for ATP resynthesis, it can be transported across the blood-brain barrier and lost from the central nervous system, or it can be metabolized to inosine and hypoxanthine. The present study used a three-dimensional in vitro cell culture model of the rat blood-brain barrier, in which forebrain astrocytes and microvascular endothelial cells were cultured in cartridges containing multiple parallel polypropylene hollow fibers. Endothelial cells were cultured on the inner surfaces and astrocytes on the outer surfaces of these fibers. Growth medium was constantly perfused through the lumen of the fibers to mimic blood flow across endothelial cells in vivo. This co-culture system was used to examine the permeation of adenosine, and its metabolite inosine, from the astrocyte compartment to the endothelial cell compartment. Dipyridamole was added to the media perfusing the endothelial cell compartment to test whether it could decrease permeation of adenosine and inosine across the in vitro blood-brain barrier. Our results indicate that dipyridamole decreased permeation of total purines, especially inosine, across the barrier. Furthermore, permeation of fluorescein isothiocyanate-labeled albumin and radiolabeled sucrose, markers of the paracellular permeation pathway, were also decreased by dipyridamole. In conclusion, these data indicate that in addition to inhibiting nucleoside efflux across the barrier, dipyridamole can also improve blood-brain barrier function in this model.

Radiofrequency Intradiscal Biacuplasty for Treatment of Discogenic Lower Back Pain: A 12-Month Follow-Up

INTRODUCTION Discogenic low back pain (LBP) affects a considerable number of patients suffering from chronic LBP. Recently, a growing interest has emerged in minimally invasive treatment options for discogenic LBP. Intradiscal biacuplasty (IDB), which uses cooled radiofrequency technology to ablate nociceptors in the posterior aspect of the intervertebral disc, is one such option. We previously presented 6-month results of a randomized, double-blinded, sham-controlled study. Now, we present the unblinded, 12-month follow-up data for treatment patients and 6-month data for cross-over subjects from the original sham group. METHODS Physical function, pain relief, and disability were assessed using the Short Form-36, numerical rating scale, and Oswestry Disability Index, respectively. Subjects were unblinded at 6 months, and those initially randomized to sham procedure were given the option to cross over to IDB. RESULTS Twenty-two out of 27 subjects in the original active treatment group were followed until 12 months and had clinically significant improvements in physical function (Δ = 22) and pain (Δ = -2.9). Out of 30 subjects originally in the sham group, 24 chose to cross over, and 20 cross-over patients completed follow-up at 6 months. In cross-over patients, improvements in physical function and pain did not differ statistically from those of patients originally randomized to IDB treatment. No complications or adverse events related to the procedure were reported. CONCLUSIONS Clinically significant improvements after IDB initially reported at 6 months were maintained at 9 and 12 months. The cross-over subjects had similar improvement in all outcome measures at all observed time points.

Adenosine permeation of a dynamic in vitro blood–brain barrier inhibited by dipyridamole

Adenosine is an inhibitory neuromodulator in the central nervous system and has been reported to have neuroprotective properties. Using a dynamic in vitro blood-brain barrier, we investigated the hypothesis that inhibition of adenosine transporters on the lumenal side of the blood-brain barrier may decrease the loss of adenosine from the brain. Our results indicate that lumenal administration of dipyridamole, a nucleoside transport inhibitor, can inhibit adenosine permeation from the extracapillary space into the lumen.

Endothelial Injury and Cell Cycle Re-Entry

Endothelial cells (ECs) affect the homeostasis of the vessel wall in terms of vasomotor tone, platelet and monocyte adhesion, growth of vascular smooth muscle cells, and extracellular matrix production. They, thereby, provide an antithrombotic and anti-inflammatory barrier for the normal vessel wall. Migration and proliferation of ECs is critical in the repair of injured vessels and in angiogenesis and vasculogenesis during development, tumor growth, and tissue repair. Regulation of the cell cycle is achieved through a complex and ordered sequence of events controlled by cyclin-dependent kinases (CDKs), the activation of which depends on regulatory phosphorylation, and their association with protein subunits, cyclins. The activation of CDKs is in turn negatively regulated by several CDK inhibitors. This chapter summarizes the most pertinent findings related to the control of cell growth and migration of vascular ECs, as relevant for maintaining the homeostasis of the vessel wall during angiogenesis and vasculogenesis, as well as for vascular remodeling during wound healing, tumor in-growth, atherogenesis, and restenosis.