Marina Frantseva

Free radical production correlates with cell death in an in vitro model of epilepsy

Free radical (FR) production, a major step in calcium‐dependent neurodegeneration, has been linked to the generation of epileptiform activity and seizure‐induced cell death. However, direct evidence of FR production in neurons during seizures has never been presented. Using hippocampal cultured slices we demonstrate that FRs are produced in CA3 but not CA1 pyramidal neurons during the rhythmic synchronous activity induced by the GABAA receptor antagonist bicuculline. The production of FRs (measured as changes in the fluorescence emission of dihydrorhodamine 123) was correlated with an increase in the baseline levels of intracellular calcium ([Ca2+]i) estimated by fluo‐3 injected into individual neurons via a patch pipette. [Ca2+]i increased during spike bursting and returned to baseline levels after the burst termination in CA1, but not in CA3, pyramidal neurons where ‘interburst’ calcium concentrations progressively increased. Measurement of cell death, performed with propidium iodide 48 h after a 30‐min exposure to bicuculline, revealed most prominent degeneration of pyramidal neurons in the CA3 pyramidal layer. The FR scavengers vitamin E and glutathione significantly reduced the seizure‐induced neurodegeneration without supressing spontaneous epileptiform activity. These observations indicate that FR overproduction is related to seizure‐induced neuronal death.

Methods to induce primary and secondary traumatic damage in organotypic hippocampal slice cultures.

Organotypic brain slice cultures have been used in a variety of studies on neurodegenerative processes [K.M. Abdel-Hamid, M. Tymianski, Mechanisms and effects of intracellular calcium buffering on neuronal survival in organotypic hippocampal cultures exposed to anoxia/aglycemia or to excitotoxins, J. Neurosci. 17, 1997, pp. 3538-3553; D.W. Newell, A. Barth, V. Papermaster, A.T. Malouf, Glutamate and non-glutamate receptor mediated toxicity caused by oxygen and glucose deprivation in organotypic hippocampal cultures, J. Neurosci. 15, 1995, pp. 7702-7711; J.L. Perez Velazquez, M.V. Frantseva, P.L. Carlen, In vitro ischemia promotes glutamate mediated free radical generation and intracellular calcium accumulation in pyramidal neurons of cultured hippocampal slices, J. Neurosci. 23, 1997, pp. 9085-9094; L. Stoppini, L.A. Buchs, D. Muller, A simple method for organotypic cultures of nervous tissue, J. Neurosci. Methods 37, 1991, pp. 173-182; R.C. Tasker, J.T. Coyle, J.J. Vornov, The regional vulnerability to hypoglycemia induced neurotoxicity in organotypic hippocampal culture: protection by early tetrodotoxin or delayed MK 801, J. Neurosci. 12, 1992, pp. 4298-4308.]. We describe two methods to induce traumatic cell damage in hippocampal organotypic cultures. Primary trauma injury was achieved by rolling a stainless steel cylinder (0.9 g) on the organotypic slices. Secondary injury was followed after dropping a weight (0.137 g) on a localised area of the organotypic slice, from a height of 2 mm. The time course and extent of cell death were determined by measuring the fluorescence of the viability indicator propidium iodide (PI) at several time points after the injury. The initial localised impact damage spread 24 and 67 h after injury, cell death being 25% and 54%, respectively, when slices were kept at 37 degrees C. To validate these methods as models to assess neuroprotective strategies, similar insults were applied to slices at relatively low temperatures (30 degrees C), which is known to be neuroprotective [F.C. Barone, G.Z. Feuerstein, R.F. White, Brain cooling during transient focal ischaemia provides complete neuroprotection, Neurosci. Biobehav. Rev. 1, 1997, pp. 31-44; V.M. Bruno, M.P. Goldberg, L.L. Dugan, R.G. Giffard, D.W. Choi, Neuroprotective effect of hypothermia in cortical cultures exposed to oxygen glucose deprivation or excitatory aminoacids, J. Neurochem. 4, 1994, pp. 387-392; G.C. Newman, H. Qi, F.E. Hospod, K. Grundhmann, Preservation of hippocampal brain slices with in vivo or in vitro hypothermia, Brain Res. 1, 1992, pp. 159-163; J.Y. Yager, J. Asseline, Effect of mild hypothermia on cerebral energy metabolism during the evolution of hypoxic ischaemic brain damage in the immature rat, Stroke, 5, 1996, pp. 919-925.]. Low temperature incubation significantly reduced cell death, now being 9% at 24 h and 14% at 67 h. Our results show that these models of moderate mechanical trauma using organotypic slice cultures can be used to study neurodegeneration and neuroprotective strategies.

Gap Junctions and Neuronal Injury: Protectants or Executioners?

The authors review concepts and recent experimental observations that relate gap junctional communication to the pathophysiology of neuronal injury, specifically ischemic or traumatic damage. The role played by this type of direct intercellular communication during the progression of the injuries can be conceived to be either detrimental or beneficial, depending on the arguments employed. The data indicate that, far from being a simple matter of judgment, the contribution of gap junctions to cell injury is a complicated phenomenon that depends on the specific insult and network in which it operates.

Development of astrocytes and neurons in cultured brain slices from mice lacking connexin43

Astrocyte and neuronal development was investigated in organotypic brain slice cultures from mouse fetuses with a null mutation in the connexin43 gene. Astrocyte morphology and electrical properties were indistinguishable in null mutant slices and control slices but at 18 days in vitro astrocyte density in the central regions of the null mutant slices was significantly higher than in control slices. Neuronal development assessed morphologically and electrophysiologically appeared normal in the mutant slices. These results suggest that intercellular communication mediated through connexin43 is not essential for the development of astrocytes and neurons but may play a role in regulating astrocytic migration.

Mitochondrial porin required for ischemia-induced mitochondrial dysfunction and neuronal damage

The precise molecular events of mitochondrial dysfunction, one of the last steps that irreversibly determines cellular degeneration and death, remain unknown. We introduce a novel strategy to isolate and assess the molecular mechanisms underlying mitochondrial dysfunction. Using an in vitro ischemia model, we obtained evidence for prolonged mitochondrial depolarization in rat organotypic hippocampal brain slices during reperfusion. Then, mitochondria were isolated from brain slices and mitochondrial proteins were purified on a cyclosporin-A affinity column. Cyclosporin-A is the most potent inhibitor of mitochondrial dysfunction, in particular the mitochondrial permeability transition, and therefore we hypothesized that it may interact with proteins involved in the permeability transition after mitochondria were subjected to manipulations that promote this event. Mitochondrial porin was reproducibly eluted from the affinity column using proteins from ischemic brain mitochondria, or from mitochondria exposed to oxidative stress that were used as a positive control. Anti-porin antibodies prevented mitochondrial depolarization and electrophysiological deterioration of hippocampal neurons during hypoxia-reperfusion, as measured by simultaneous fluorescence imaging and whole-cell recordings. These observations provide biochemical and functional evidence that porin is directly involved in mitochondrial dysfunction and neuronal impairment during ischemia-reperfusion, and indicate that porin could be a novel therapeutic target to prevent cellular degeneration.

Neurotrauma/neurodegeneration and mitochondrial dysfunction.

Publisher Summary This chapter focuses on neurodegeneration and mitochondrial dysfunction. The chapter presents the in vitro model of ischemic injury, which consists of superfusing organotypic cultured hippocampal slices with glucose-free deoxygenated solution for 8 min. After an ischemic episode, free radicals are produced in pyramidal neurons mostly during reoxygenation and intracellular calcium levels increase in parallel to free radical generation. The chapter presents evidence for a prolonged mitochondrial depolarization, indicative of the mitochondrial permeability transition (MPT), during reperfusion in organotypic hippocampal neurons, by using two mitochondrial dyes: rhodamine 123 and JC-1. Mitochondrial dysfunction results from several converging deleterious mechanisms. Mitochondrial dysfunction also plays a major role in traumatic cell death. To date mitochondria have been little studied for their involvement in traumatic brain injury, but they could be an important therapeutic target for the early treatment and prevention of secondary brain and spinal cord damage.

Anti-Porin Antibodies Prevent Excitotoxic and Ischemic Damage to Brain Tissue

The mitochondrial permeability transition (MPT) is a converging event for different molecular routes leading to cellular death after excitotoxic/oxidative stress, and is considered to represent the opening of a pore in the mitochondrial membrane. There is evidence that the outer mitochondrial membrane protein porin is involved in the MPT and apoptosis. We present here a proof-of-principle study to address the hypothesis that anti-porin antibodies can prevent excitotoxic/ischemia-induced cell death. We generated anti-porin antibodies and show that the F(ab)(2) fragments penetrate living cells, reduce Ca(2+)-induced mitochondrial swelling as other MPT blockers do, and decrease neuronal death in dissociated and organotypic brain slice cultures exposed to excitotoxic and ischemic episodes. These observations present direct evidence that anti-porin antibody fragments prevent cell damage in brain tissue, that porin is a crucial protein involved in mitochondrial and cell dysfunction, and that it is conceivable that antibodies can be used as therapeutic agents.

Molecular Mechanisms of Free Radical Production and Protective Efficacies of Antioxidants in in Vitro Ischemia-Reperfusion

Compelling evidence implicates free radicals (FRs) as major contributors to ischemic and excitotoxic tissue injury in the CNS.1 The biochemical mechanisms leading to FR production during ischemic brain injury remain unclear owing to methodological difficulties in detecting short-lived FRs in in vivo experiments, and a lack of neuronal circuitry required for FR generation in most of the in vitro model systems. We investigated the mechanisms of FR overproduction in rat CA1 pyramidal neurons of organotypic hippocampal slices during ischemia-reperfusion injury (IRI). IRI was initiated by superfusion of cultured hippocampal slices with glucose-free deoxygenated artificial cerebrospinal fluid (ACSF) for 8 minutes, as described in detail in reference 2. Ischemia-induced free radical generation (measured as changes in the fluorescence emission of dihydrorhodamine123) temporally correlated with intracellular calcium elevation, as measured by injection of fluo-3 in individual pyramidal cells employing patch electrodes. Both FR generation and intracellular calcium accumulation were markedly diminished in the presence of glutamate receptor blockers AP-5 and CNQX, implicating glutamate-mediated [Ca]i rises as a direct cause of FR overproduction resulting from IRI. FR generation was greatly decreased by the mitochondrial complex I blocker, rotenone, indicating that mitochondria are the principal source of ischemic FR production. Measurements of mitochondrial calcium with the mitochondrial calcium probe rhod-2, revealed that peaks of FR production during and after the anoxic episode always followed the step-like increases of mitochondrial calcium, suggesting that mitochondrial calcium overload is linked to ischemic mitochondrial FR generation. Biochemical cascades initiated by oxidative stress and excitotoxic intracellular calcium rises are thought to converge on mitochondrial dysfunction (mitochondrial permeability transition3). The mitochondrial permeability transition (MPT) is a gradual loss of mitochondrial potential accompanied by the swelling of the organelles, prevented by cyclosporine A (CsA).3 A gradual, CsA-sensitive decrease of the mitochondrial potential, indicative of the MPT, has been previously observed in our ischemia model.4 Because the MPT resulting from oxidative stress has been shown to cause secondary FR generation in hepatocytes,5 we sought to investigate the possible contribution of IRI-induced mitochondrial dysfunction to FR genera-

Mitochondrial porin, a novel target to prevent ischemia-induced neurodegeneration?

The mitochondrial permeability transition (MPT) is considered to represent one of the final events that results in irreversible damage and subsequent cell death.1 The MPT has been described in a variety of cell systems and occurs as a consequence of oxidative insults and excitotoxicity.2 Recent reports indicate that the permeability transition could also occur in neurons, since it has been inferred from experiments using isolated brain mitochondria in the presence of elevated calcium and in dissociated neuronal or glial cultures exposed to glutamate or N-methyl-D-aspartate.3–6 Using an in vitro ischemia model, we obtained evidence for prolonged mitochondrial depolarization in rat organotypic hippocampal brain slices during reperfusion. Techniques for culturing embryonic brain slices were prepared as described previously.7 The experiments were carried out after 7–14 days in vitro. Hypoxia-hypoglycemia was initiated by superfusing slices for 8 minutes with glucose-free artificial cerebrospinal fluid (ACSF) aerated with 95% N2/5% CO2, sucrose (10 mM) was added to the solution to maintain osmolarity.7 The organotypic hippocampal cultures were loaded with the mitochondrial dye rhodamine123 (R123, 15 μM), which reflects the mitochondrial potential and is used as an indicator of the MPT. R123 fluorescence increased briefly in CA1 neurons for 1–2 minutes before becoming diffuse and disappearing abruptly during reperfusion in 5 of 6 slices, or during the ischemic insult (1 of 6). Similar results were obtained when slices were loaded with the ratiometric mitochondrial dye JC-1 (n = 4). The loss of mitochondrial potential in CA1 neurons during hypoxia-hypoglycemia and reperfusion was prevented by cyclosporin-A (CsA, n = 4). CsA is also a potent blocker of the MPT1 and prevents mitochondrial depolarization induced by N-methyl-D-aspartate.3 We then used a novel strategy to identify the molecular mechanisms underlying mitochondrial dysfunction, based on CsA-affinity chromatography of mitochondrial proteins from injured and control brain tissue. Mitochondria were isolated from brain slices and mitochondrial proteins were purified on a CsA affinity column.8 CsA is the most potent inhibitor of mitochondrial dysfunction, in particular the MPT,9 and therefore we hypothesized that it may interact with proteins involved in the permeability transition after mitochondria were subjected to manipulations that promote the MPT. Mitochondrial porin was specifically eluted with 0.42 mM CsA from the affinity column using proteins from ischemic brain mitochondria (n = 5),