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Cell-permeant Ca2+ chelators reduce early excitotoxic and ischemic neuronal injury in vitro and in vivo
Abstract
We report the characterization of the first successful treatment of neuronal ischemic injury in vivo by cell-permeant Ca2+ chelators. The chelators attenuated glutamate-induced intracellular Ca2+ increases and neurotoxicity in neuronal explant cultures. When infused intravenously in rats, permeant fluorescent BAPTA analogs accumulated in neurons in several brain regions. BAPTA-AM, infused in vivo, reduced Ca(2+)-dependent spike frequency adaptation and post-spike train hyperpolarizations in CA1 neurons taken from treated animals. This effect was reproduced by direct injections of BAPTA into untreated neurons. The effects of three different chelators (BAPTA, 5,5'-difluoro BAPTA, and 4,4'-difluoro BAPTA) on Ca(2+)-dependent membrane excitability varied with their Ca2+ affinity. When the chelators' permeant forms were used to treat rats prior to the induction of focal cortical ischemia, they were highly neuroprotective, as gauged by significant reductions in cortical infarction volumes and neuronal sparing. The chelators' protective effects in vivo also reflected their affinity for Ca2+. This report provides the most direct evidence to date that intracellular Ca2+ excess triggers early neurodegeneration in vivo and contributes a novel therapeutic approach to neuronal ischemia of potential clinical utility.
... The reason is the large amount of Glu diffused from dead neurons and released from astrocytes due to the reversal of glutamate transporters (Dirnagl et al., 1999;Allen et al., 2004;Malarkey and Parpura, 2008). Longterm exposure to high doses of Glu causes in neuronal cultures a biphasic increase in [Ca 2+ ] i (Tymianski et al., 1993;Adamec et al., 1998;Castilho et al., 1998). The second phase of the rise of [Ca 2+ ] i , the so-called delayed calcium deregulation (DCD), is always synchronized with a profound drop in the mitochondrial transmembrane potential (Vergun et al., 1999;Khodorov, 2004). ...
... The solution was changed by a triple washout with a new solution within <25 s. For partial depolarization of mitochondria and verification of the Ca 2+ uptake in the presence of Glu, mitochondria were depolarized by application of protonophore carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) (Tymianski et al., 1993;Nicholls and Budd, 2000). ...
Lipopolysaccharide (LPS), a fragment of the bacterial cell wall, specifically interacting with protein complexes on the cell surface, can induce the production of pro-inflammatory and apoptotic signaling molecules, leading to the damage and death of brain cells. Similar effects have been noted in stroke and traumatic brain injury, when the leading factor of death is glutamate (Glu) excitotoxicity too. But being an amphiphilic molecule with a significant hydrophobic moiety and a large hydrophilic region, LPS can also non-specifically bind to the plasma membrane, altering its properties. In the present work, we studied the effect of LPS from Escherichia coli alone and in combination with the hyperstimulation of Glu-receptors on the functional state of mitochondria and Ca ²⁺ homeostasis, oxygen consumption and the cell survival in primary cultures from the rats brain cerebellum and cortex. In both types of cultures, LPS (0.1–10 μg/ml) did not change the intracellular free Ca ²⁺ concentration ([Ca ²⁺ ] i ) in resting neurons but slowed down the median of the decrease in [Ca ²⁺ ] i on 14% and recovery of the mitochondrial potential (ΔΨm) after Glu removal. LPS did not affect the basal oxygen consumption rate (OCR) of cortical neurons; however, it did decrease the acute OCR during Glu and LPS coapplication. Evaluation of the cell culture survival using vital dyes and the MTT assay showed that LPS (10 μg/ml) and Glu (33 μM) reduced jointly and separately the proportion of live cortical neurons, but there was no synergism or additive action. LPS-effects was dependent on the type of culture, that may be related to both the properties of neurons and the different ratio between neurons and glial cells in cultures. The rapid manifestation of these effects may be the consequence of the direct effect of LPS on the rheological properties of the cell membrane.
... That this is so has been particularly well demonstrated by the experiments of Tymianski et al. (1993) Ikonomidou et al., 1989) while sensitivity to kainate is low (Campochiaro and Coyle, 1978). In contrast, there is a high sensitivity to kainate in the adult rat brain and NMDA produces little neurotoxicity (Olney, 1978). ...
... This possibility is supported by single channel recordings that show kainate can activate conductances similar to that of NMDA but with lower probability Usowicz, 1987, Jahr andStevens, 1987 The neurotoxic effects of glutamate and its analogues are generally thought to involve an increase in [Ca^^]i which then triggers cell death (Rothman, 1983(Rothman, , 1984. Tymianski et al. (1993) (Yin et aL, 1994, Brorson et al., 1994. et al., , Ikonomidou et al., 1989. ...
Neurotransmitters exert important influences on a variety of processes during the development of the central nervous system, including neurite outgrowth, cell migration and cell survival. In this thesis the embryonic chick retina has been used to investigate the temporal sequence in which neurotransmitter receptors develop, their influence on intracellular calcium concentration ([Ca2+]i) and their possible functions during development. Electrophysiological and Ca2+ imaging techniques show that receptors for the excitatory neurotransmitters glutamate and acetylcholine and for the inhibitory neurotransmitter GABA appear prior to synaptogenesis, suggesting possible developmental roles for these transmitters. Many of the developmental effects of glutamate and other transmitters are attributed to their ability to alter [Ca2+]i and recently it has been shown that AMPA/kainate receptors that exclude the GLUR2 subunit are Ca2+-permeable. Here the presence of Ca2+-permeable AMPA/kainate receptors in the embryonic chick retina is demonstrated both in vivo and in vitro. Retinal explants and cultures of dissociated retinal cells were treated with glutamate analogues to investigate the role of non-NMDA (AMPA/kainate) receptors during the development of the chick retina. Activation of AMPA/kainate receptors with kainate late in retinal development leads to excitotoxic cell death, however exposure to this neurotoxin early on has no affect on cell survival in both dissociated cultures and explant cultures. Cell survival in kainate is correlated with a decrease in the Ca2+-permeability of the AMPA/kainate receptor, suggesting it may play a role in cell survival during development. Activation of this receptor early in development produces a significant reduction in the number of processes extended by the cells, showing glutamate may influence neurite growth in the developing retina via activation of non-NMDA receptors.
... To examine nuclear mechanosensing-triggered pathways during homeostatic SMG surveillance, we used two-photon intravital microscopy (2PM) to quantify baseline OT-I T RM motility parameters in LCMV-OVA-immunized SMGs before overlaying 20 or 50 μM BAPTA-AM on the SMG preparation for 20 min and recording T RM motility for additional 1 to 4 hours (Fig. 6B and movie S15). BAPTA-AM has been reported to accumulate efficiently in cells in vivo (64). Accordingly, the short BAPTA-AM superfusion caused a dose-dependent decrease in SMG T RM speeds and directionality (Fig. 6, C to F), with a concomitant increase in arrest coefficients and sphericity (Fig. 6, F and G). ...
Tissue-resident CD8 ⁺ T cells (T RM ) continuously scan peptide-MHC (pMHC) complexes in their organ of residence to intercept microbial invaders. Recent data showed that T RM lodged in exocrine glands scan tissue in the absence of any chemoattractant or adhesion receptor signaling, thus bypassing the requirement for canonical migration-promoting factors. The signals eliciting this noncanonical motility and its relevance for organ surveillance have remained unknown. Using mouse models of viral infections, we report that exocrine gland T RM autonomously generated front-to-back F-actin flow for locomotion, accompanied by high cortical actomyosin contractility, and leading-edge bleb formation. The distinctive mode of exocrine gland T RM locomotion was triggered by sensing physical confinement and was closely correlated with nuclear deformation, which acts as a mechanosensor via an arachidonic acid and Ca ²⁺ signaling pathway. By contrast, naïve CD8 ⁺ T cells or T RM surveilling microbe-exposed epithelial barriers did not show mechanosensing capacity. Inhibition of nuclear mechanosensing disrupted exocrine gland T RM scanning and impaired their ability to intercept target cells. These findings indicate that confinement is sufficient to elicit autonomous T cell surveillance in glands with restricted chemokine expression and constitutes a scanning strategy that complements chemosensing-dependent migration.
... On the other hand, chelating calcium had no significant effect on the TAU missorting induced by mitochondrial impairment in our experiments. Although this mechanism of neuroprotection has been shown before for other neuronal injuries [50,51], these injuries also led to a higher increase in calcium than our mitochondrial impairment which might be the cause that the protective effect was more pronounced there. Therefore, while both elevated calcium and microtubule impairment are tightly linked to TAU missorting and so far the two most discussed triggers [4,52], in our case impaired microtubule function/dynamics may be causative or at least the major factor for mitochondrial impairmentinduced TAU missorting. ...
Loss of neuronal polarity and missorting of the axonal microtubule-associated-protein TAU are hallmarks of Alzheimer’s disease (AD) and related tauopathies. Impairment of mitochondrial function is causative for various mitochondriopathies, but the role of mitochondria in tauopathies and in axonal TAU-sorting is unclear. The axon-initial-segment (AIS) is vital for maintaining neuronal polarity, action potential generation, and—here important—TAU-sorting. Here, we investigate the role of mitochondria in the AIS for maintenance of TAU cellular polarity. Using not only global and local mitochondria impairment via inhibitors of the respiratory chain and a locally activatable protonophore/uncoupler, but also live-cell-imaging and photoconversion methods, we specifically tracked and selectively impaired mitochondria in the AIS in primary mouse and human iPSC-derived forebrain/cortical neurons, and assessed somatic presence of TAU. Global application of mitochondrial toxins efficiently induced tauopathy-like TAU-missorting, indicating involvement of mitochondria in TAU-polarity. Mitochondria show a biased distribution within the AIS, with a proximal cluster and relative absence in the central AIS. The mitochondria of this cluster are largely immobile and only sparsely participate in axonal mitochondria-trafficking. Locally constricted impairment of the AIS-mitochondria-cluster leads to detectable increases of somatic TAU, reminiscent of AD-like TAU-missorting. Mechanistically, mitochondrial impairment sufficient to induce TAU-missorting results in decreases of calcium oscillation but increases in baseline calcium, yet chelating intracellular calcium did not prevent mitochondrial impairment-induced TAU-missorting. Stabilizing microtubules via taxol prevented TAU-missorting, hinting towards a role for impaired microtubule dynamics in mitochondrial-dysfunction-induced TAU-missorting. We provide evidence that the mitochondrial distribution within the proximal axon is biased towards the proximal AIS and that proper function of this newly described mitochondrial cluster may be essential for the maintenance of TAU polarity. Mitochondrial impairment may be an upstream event in and therapeutic target for AD/tauopathy.
... It had been reported that cortical neurons occurred apoptosis when the concentration of glutamate was 30~100 μmol/l, while necrosis took place in cortical neurons when the concentration was 300 μmol/l (Yu et al., 2003). Large amounts of glutamate are released as neurotoxic substances from ischemic cerebral tissue (Tymianski et al., 1993). Although touching with glutamate transitorily, cultured neurons rapidly get swelling due to increasing Na + permeability which leads to water across the cellular membrane into intracellular. ...
Background
: Ischemic stroke is a common cerebrovascular disease. Due to sudden interruption of blood flow by arterial thrombus, amounts of neurons in ischemic central and penumbral regions occur necrosis and apoptosis resulting in serious injury of neurological function. Chinese medicines have a great advantage in ischemic stroke treatment and recovery, especially Angelica sinensis.
Purpose
: There are a large number of studies reported that Angelica injection and A. sinensis active compounds. We systematically reviewed the effects and mechanisms of A. sinensis in recent years according to the guidelines of the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) statements, and excavated its therapeutic potentiality for exploring more effective and safe compounds for ischemic stroke precision treatment.
Results
: A. sinensis extracts and active compounds, such as Z-ligustilide, 3-n-Butylphthalide, and ferulic acid have significant effects of anti-inflammation, anti-oxidative stress, angiogenesis, neurogenesis, anti-platelet aggregation, anti-atherosclerosis, protection of vessels, which contributes to improvement of neurological function on ischemic stroke.
Conclusion
: A. sinensis is a key agent for ischemic stroke treatment, and worth deeply excavating its therapeutic potentiality with the aid of pharmacological network, computer-aided drug design, artificial intelligence, big data and multi-scale modelling techniques.
... As proof of this, a significant protection against neuronal death has been demonstrated by the direct antagonism of NMDA and AMPA receptors [5]. Their neuroprotective mechanism could work by antagonizing the cellular calcium influx [8] or through a chelating effect on intracellular calcium [9]. ...
One of the causes of nervous system degeneration is an excess of glutamate released upon several diseases. Glutamate analogs, like N-methyl-DL-aspartate (NMDA) and kainic acid (KA), have been shown to induce experimental retinal neurotoxicity. Previous results have shown that NMDA/KA neurotoxicity induces significant changes in the full field electroretinogram response, a thinning on the inner retinal layers, and retinal ganglion cell death. However, not all types of retinal neurons experience the same degree of injury in response to the excitotoxic stimulus. The goal of the present work is to address the effect of intraocular injection of different doses of NMDA/KA on the structure and function of several types of retinal cells and their functionality. To globally analyze the effect of glutamate receptor activation in the retina after the intraocular injection of excitotoxic agents, a combination of histological, electrophysiological, and functional tools has been employed to assess the changes in the retinal structure and function. Retinal excitotoxicity caused by the intraocular injection of a mixture of NMDA/KA causes a harmful effect characterized by a great loss of bipolar, amacrine, and retinal ganglion cells, as well as the degeneration of the inner retina. This process leads to a loss of retinal cell functionality characterized by an impairment of light sensitivity and visual acuity, with a strong effect on the retinal OFF pathway. The structural and functional injury suffered by the retina suggests the importance of the glutamate receptors expressed by different types of retinal cells. The effect of glutamate agonists on the OFF pathway represents one of the main findings of the study, as the evaluation of the retinal lesions caused by excitotoxicity could be specifically explored using tests that evaluate the OFF pathway.
... The concentration of BAPTA was determined considering previous studies. [23][24][25] The BAPTA or DPBS were treated in drops on the full-transection spot with pipette and were arranged into BAPTA group and vehicle control group, respectively. The muscle and fascia were sutured, and the skin was closed. ...
Aim
Despite animal evidence of a role of calcium in the pathogenesis of spinal cord injury, several studies conducted in the past found calcium blockade ineffective. However, those studies involved oral or parenteral administration of Ca++ antagonists. We hypothesized that Ca++ blockade might be effective with local/immediate application (LIA) at the time of neural injury.
Methods
In this study, we assessed the effects of LIA of BAPTA (1,2‐bis (o‐aminophenoxy) ethane‐N, N, N′, N'‐tetraacetic acid), a cell‐permeable highly selective Ca++ chelator, after spinal cord transection (SCT) in mice over 4 weeks. Effects of BAPTA were assessed behaviorally and with immunohistochemistry. Concurrently, BAPTA was submitted for the first time to multimodality assessment in an in vitro model of neural damage as a possible spinal neuroprotectant.
Results
We demonstrate that BAPTA alleviates neuronal apoptosis caused by physical damage by inhibition of neuronal apoptosis and reactive oxygen species (ROS) generation. This translates to enhanced preservation of electrophysiological function and superior behavioral recovery.
Conclusion
This study shows for the first time that local/immediate application of Ca++ chelator BAPTA is strongly neuroprotective after severe spinal cord injury.
... Calcium (Ca 2+ ) imbalance is a significant trigger that leads to neuronal cell death during brain damage, and its inhibition directly correlates with ischemic neuroprotection (Berliocchi et al., 2006;Choi, 1995;Tymianski et al., 1993). Different synaptic preparation studies have shown that Ca 2+ can accelerate or inhibit endocytosis at nerve terminals (Balaji et al., 2008;Wu et al., 2009Yao and Sakaba, 2012); nevertheless, the precise steps by which Ca 2+ acts in this process have not been identified. ...
We first explore the features of GluK2 endocytosis during kainate excitotoxicity and then explore the role of Ca2+ in the regulation of GluK2 endocytosis. The roles of Ca2+ were examined by treating cells with Ca2+ inhibitors or chelators. Surface biotinylation was used to examine the surface localization of GluK2. Immunoprecipitation followed by immunoblotting was used to identify the interaction of GluK2 with the endocytosis regulator protein-interacting with C kinase 1 and dynamin. Dynamin phosphorylation was examined by immunoblotting with the corresponding antibodies. Our results show that GluK2 internalization is blocked by inhibitors of clathrin-independent endocytosis and relies on intracellular Ca2+/calcineurin signaling. Protein-interacting with C kinase 1-GluK2 interaction is regulated by Ca2+/calcineurin signaling. Dynamin participates in the regulation of GluK2 surface localization. Also, calcineurin activation is related to dynamin function during kainate excitotoxicity. In conclusion, GluK2 receptor endocytosis is probably a clathrin-independent and dynamin-dependent process regulated by the peak Ca2+ transient. This work indicates the roles of the Ca2+ network in the regulation of GluK2 endocytosis during kainate excitotoxicity.
... Several mechanisms are responsible for up-regulation of Bcl2 in cells, one of the chromosomal translocation (14;18) that places the Bcl2 gene next to enhancer element of the immunoglobulin heavy chain promoter [130,131] which is a central mechanism for increased expression of Bcl2. In addition to this, Bcl2 gene rearrangement [132] and hypomethylation [133], loss of miR15a and miR16 which repress the expression of Bcl2 mRNA, responsible for 50% of chronic lymphocytic leukaemia (CLL) due to chromosome deletion and mutation [134]. Overexpression of other Bcl2regulating miRNAs (miR-195, miR-204, miR-24-2 and miR-365-2), significantly enhanced the efficiency of chemotherapy in lung, colon cancers and breast [135,136]. ...
Apoptosis, a well-characterized and regulated cell death programme in eukaryotes plays a fundamental role in developing or later-life periods to dispose of unwanted cells to maintain typical tissue architecture, homeostasis in a spatiotemporal manner. This silent cellular death occurs without affecting any neighboring cells/tissue and avoids triggering of immunological response. Furthermore, diminished forms of apoptosis result in cancer and autoimmune diseases, whereas unregulated apoptosis may also lead to the development of a myriad of neurodegenerative diseases. Unraveling the mechanistic events in depth will provide new insights into understanding physiological control of apoptosis, pathological consequences of abnormal apoptosis and development of novel therapeutics for diseases. Here we provide a brief overview of molecular players of programmed cell death with discussion on the role of caspases, modifications, ubiquitylation in apoptosis, removal of the apoptotic body and its relevance to diseases.
... Next, we directly examined the role of presynaptic Ca 2+ on neurotransmission in HD cortical neurons by loading neurons with the membrane-permeable Ca 2+ chelator BAPTA-AM (Tsien, 1981) and measuring vesicle fusion in single FM 1-43-loaded presynaptic terminals. We used 200 nM BAPTA for these experiments, as this chelator has been reported to protect neurons against excitotoxicity (Tymianski et al., 1993(Tymianski et al., , 1994Abdel-Hamid and Tymianski, 1997). As shown in Figure 5A, HD cortical neurons loaded with BAPTA-AM had significantly reduced the release of vesicles compared to control HD neurons (loaded with DMSO); this reduction was reflected in both the significant average fluorescence loss [37.9 ± 2.0% (n = 32 boutons, N = 6 experiments with BAPTA-AM) vs. 57.8 ...
Huntington’s disease (HD) is an inherited neurodegenerative disorder caused by the abnormal expansion of CAG repeats in the huntingtin (HTT) gene, which leads to progressive loss of neurons starting in the striatum and cortex. One possible mechanism for this selective loss of neurons in the early stage of HD is altered neurotransmission at synapses. Despite the recent finding that presynaptic terminals play an important role in HD, neurotransmitter release at synapses in HD remains poorly understood. Here, we measured synaptic vesicle release in real time at single presynaptic terminals during electrical field stimulation. We found the increase in synaptic vesicle release at presynaptic terminals in primary cortical neurons in a knock-in mouse model of HD (zQ175). We also found the increase in Ca²⁺ influx at presynaptic terminals in HD neurons during the electrical stimulation. Consistent with increased Ca²⁺-dependent neurotransmission in HD neurons, the increase in vesicle release and Ca²⁺ influx was rescued with Ca²⁺ chelators or by blocking N-type voltage-gated Ca²⁺ channels, suggesting N-type voltage-gated Ca²⁺ channels play an important role in HD. Taken together, our results suggest that the increased synaptic vesicles release due to increased Ca²⁺ influx at presynaptic terminals in cortical neurons contributes to the selective neurodegeneration of these neurons in early HD and provide a possible therapeutic target.
... This approach stands in contrast to screening protocols in which functional inducer and small molecule therapeutic are tested coincident with one another, frequently identifying candidate compounds which ultimately fail in real clinical settings. 23 25,26 However, the precise molecular mechanism underlying these observations remains unclear. ...
Aberrant regulation of programmed cell death (PCD) has been tied to an array of human pathologies ranging from cancers to autoimmune disorders to diverse forms of neurodegeneration. Pharmacologic modulation of PCD signalling is therefore of central interest to a number of clinical and biomedical applications. A key component of PCD signalling involves the modulation of pro-and anti-apoptotic Bcl-2 family members. Among these, Bax translocation represents a critical regulatory phase in PCD. In the present study, we have employed a high-content high-throughput screen to identify small molecules which inhibit the cellular process of Bax re-distribution to the mitochondria following commitment of the cell to die. Screening of 6246 Generally Recognized As Safe compounds from four chemical libraries post-induction of cisplatin-mediated PCD resulted in the identification of 18 compounds which significantly reduced levels of Bax translocation. Further examination revealed protective effects via reduction of executioner caspase activity and enhanced mito-chondrial function. Consistent with their effects on Bax translocation, these compounds exhibited significant rescue against in vitro and in vivo cisplatin-induced apoptosis. Altogether, our findings identify a new set of clinically useful small molecules PCD inhibitors and highlight the role which cAMP plays in regulating Bax-mediated PCD.
... Moreover, BAPTA-AM has been reported to protect neurons in the experimental model of focal cerebral ischemia. 25 26 Nevertheless, there is no research to explore the effect of BA-N on I/R induced injury in rat kidneys. With the development of nanotechnology and nanomedicine, nanoparticles (NPs) have received increasing attention as drug carriers in research. ...
Ischemia-reperfusion (I/R) is a major cause of acute kidney injury (AKI), which is associated with unacceptably high mortality rates in ICU. This research was designed to explore the therapeutic effect of BAPTA-AM (1, 2-Bis(2-aminophenoxy) ethane-N, N, N, N-tetraacetic acid tetrakis(acetoxymethyl ester)) nanoparticle (BA-N) on AKI. BA-N was developed by liposome strategy and characterized by standard methods. The rat model was selected and the rats were randomly allocated into four groups: (1) Normal group; (2) Sham-operated group; (3) Model group (I/R+NS); (4) BA-N treatment group (I/R+BA-N). AKI model was established via clipping the bilateral renal artery with a microvascular clamp for 45 min. After reperfusion, serum cystatin C (Cys C), creatinine (Cr), blood urea nitrogen (BUN), lactate dehydrogenase (LDH) and caspase 3 levels were determined for the assessment of renal function. Kidney samples were then collected for the measurement of renal malondialdehyde (MDA) level and superoxide dismutase (SOD) activity. The assays of histological examination, ELISA, immunohistochemistry, western blot, TUNEL and RT-PCR were utilized for the detection of apoptosis. The results demonstrated that AKI model caused a significant decreasing in SOD activity, accompanied by a remarkable increase in Cys C, Cr, BUN, LDH, MDA, caspase 3 and cytochrome c (Cyt C) level, compared to the control group. BA-N (100 μg/kg i.v.) significantly improved renal function and histopathological appearance, restored MDA level and SOD activity, decreased Bax/Bcl-2 ratio, caspase 3 activity, Cyt C release and TUNEL positive apoptotic cells. Our studies indicated that BA-N plays a renal-protective role, probably through antiapoptotic and antioxidant mechanisms. BA-N may regulate mitochondria pathway via decreasing Bax/Bcl-2 ratio, inhibiting caspase 3 expression and Cyt C release. Overall, BA-N may have potentials as an anti-AKI drug.
... It has been reported that the endogenous buffer capacity of central neurons is increased during maturation and increase in its buffer capacity can rescue the cognitive deficits 52 . Our observation that high concentration of EGTA in cytoplasm can prevent enhancement of calcium currents from bilirubin is consistent with the observation that Ca 2+ chelators may protect neurons from early neurodegeneration triggered by excess intracellular Ca 2+ in vivo 53,54 . Residual Ca 2+ would prolong Ca 2+ channel opening, and enhance facilitation 11 . ...
Neonatal brain is particularly vulnerable to pathological levels of bilirubin which elevates and overloads intracellular Ca2+, leading to neurotoxicity. However, how voltage-gated calcium channels (VGCCs) are functionally involved in excess calcium influx remains unknown. By performing voltage-clamp recordings from bushy cells in the ventral cochlear nucleus (VCN) in postnatal rat pups (P4-17), we found the total calcium current density was more than doubled over P4-17, but the relative weight of VGCC subtypes changed dramatically, being relatively equal among T, L, N, P/Q and R-type at P4-6 to predominantly L, N, R over T and P/Q at P15-17. Surprisingly, acute administration of bilirubin augmented the VGCC currents specifically mediated by high voltage-activated (HVA) P/Q-type calcium currents. This augment was attenuated by intracellular loading of Ca2+ buffer EGTA or calmodulin inhibitory peptide. Our findings indicate that acute exposure to bilirubin increases VGCC currents, primarily by targeting P/Q-type calcium channels via Ca2+ and calmodulin dependent mechanisms to overwhelm neurons with excessive Ca2+. Since P/Q-subtype calcium channels are more prominent in neonatal neurons (e.g. P4-6) than later stages, we suggest this subtype-specific enhancement of P/Q-type Ca2+ currents likely contributes to the early neuronal vulnerability to hyperbilirubinemia in auditory and other brain regions.
... Other mechanisms include BCL2 gene rearrangement 144 and hypomethylation. 145 In addition, loss of miR-15a and miR-16, which target and repress BCL2 mRNA, occurs in more than 50% of chronic lymphocytic leukemia (CLL) as a result of chromosome deletions and mutations. 146 Conversely, overexpression of other BCL2-regulating miRNAs, including miR-195, miR-24-2, miR-365-2, and miR-204, reportedly increases the efficiency of chemotherapy in breast, 147 lung, and colon cancers. ...
Apoptosis is a morphologically and biochemically distinct form of cell death that plays an essential role in development, immune response, and tissue homeostasis. Diminished apoptosis is also considered a hallmark of cancer whereas many cancer treatments induce apoptosis in susceptible cells. Classically, this induction of apoptosis occurs through two major signaling pathways: the extrinsic pathway and the intrinsic pathway. It has been known for 20 years that B-cell lymphoma-2 (BCL2) family proteins control the intrinsic apoptotic pathway by regulating the process of mitochondrial outer membrane permeabilization (MOMP) through protein-protein interactions. Recent studies have elucidated how BCL2 antagonist/killer (BAK) and BCL2-associated X protein (BAX) are activated by BCL2 homology 3 (BH3)-only proteins and how activated BAK and BAX permeabilize MOM, providing increased understanding of how BCL2 family proteins control MOMP. Moreover, both structural and biochemical studies have revealed dual roles for anti-apoptotic BCL2 family proteins in inhibiting BH3-only proteins and restraining activated BAK and BAX. Here, we review recent advances in understanding how BCL2 family proteins control MOMP as well as new nonapoptotic functions for these proteins.
... Neuroscientists and clinicians have known for many decades that perikaryal survival decreases for neurons whose axons or dendrites are transected nearer to the soma, compared to further from the soma (Ramón y Cajal, 1928;Lucas et al., 1985Lucas et al., , 1990Yoo et al., 2004;Nguyen et al., 2005;Campbell, 2008;Wolfe et al., 2010). Ca 2+ influx at cut axonal ends increases somal Ca 2+ concentrations that probably lead to the disruption of somal protein synthesis, and trigger various pathways that can lead to cellular apoptosis (Choi, 1988;Tymianski et al., 1993;Ziv and Spira, 1995;Yoo et al., 2004;Nguyen et al., 2005). ...
The repair (sealing) of plasmalemmal damage, consisting of small holes to complete transections, is critical for cell survival, especially for neurons that rarely regenerate cell bodies. We first describe and evaluate different measures of cell sealing. Some measures, including morphological/ultra-structural observations, membrane potential, and input resistance, provide very ambiguous assessments of plasmalemmal sealing. In contrast, measures of ionic current flow and dye barriers can, if appropriately used, provide more accurate assessments. We describe the effects of various substances (calcium, calpains, cytoskeletal proteins, ESCRT proteins, mUNC-13, NSF, PEG) and biochemical pathways (PKA, PKC, PLC, Epac, cytosolic oxidation) on plasmalemmal sealing probability, and suggest that substances, pathways, and cellular events associated with plasmalemmal sealing have undergone a very conservative evolution. During sealing, calcium ion influx mobilizes vesicles and other membranous structures (lysosomes, mitochondria, etc.) in a continuous fashion to form a vesicular plug that gradually restricts diffusion of increasingly smaller molecules and ions over a period of seconds to minutes. Furthermore, we find no direct evidence that sealing occurs through the collapse and fusion of severed plasmalemmal leaflets, or in a single step involving the fusion of one large wound vesicle with the nearby, undamaged plasmalemma. We describe how increases in perikaryal calcium levels following axonal transection account for observations that cell body survival decreases the closer an axon is transected to the perikaryon. Finally, we speculate on relationships between plasmalemmal sealing, Wallerian degeneration, and the ability of polyethylene glycol (PEG) to seal cell membranes and rejoin severed axonal ends – an important consideration for the future treatment of trauma to peripheral nerves. A better knowledge of biochemical pathways and cytoplasmic structures involved in plasmalemmal sealing might provide insights to develop treatments for traumatic nerve injuries, stroke, muscular dystrophy, and other pathologies.
... Notably, calcium accumulation occurs before and is a strong predictor of neuronal death following cerebral ischemia (Deshpande et al., 1987). Thus, protection against neuronal hypoxia in vitro and cerebral ischemia in vivo can be achieved by direct NMDAR antagonism (Goldberg et al., 1987;Rothman et al., 1987;Simon et al., 1984;Weiss et al., 1986), antagonism of the AMPA receptor, which is required for NMDAR activation (Sheardown et al., 1990), simultaneous antagonism of both the NMDAR and AMPA receptor (Germano et al., 1987;Goldberg et al., 1987;Rothman, 1984;Simon et al., 1986), antagonism of the L-type calcium channels, which can contribute to calcium overload (Uematsu et al., 1989), and intracellular calcium chelation (Tymianski et al., 1993c). In general, direct NMDAR antagonism appears to be sufficiently neuroprotective against hypoxic neuronal death compared to co-antagonism of different excitotoxic receptors (Goldberg and Choi, 1993;Goldberg et al., 1987) and calcium sources (see Section 2.2). ...
... Neuroscientists and clinicians have known for many decades that transection of dendrites or axons can initiate a Ca 2+ influx that leads to perikaryial death (Ramón y Cajal, 1928;Schlaepfer and Bunge, 1973;Lucas et al., 1985Lucas et al., , 1990Loewy and Schader, 1977;Yoo et al., 2004;Nguyen et al., 2005;Campbell, 2008;Wolfe et al., 2010;Moe et al., 2015). This influx increases somal Ca 2+ concentrations, activating calpains and other proteases in enzymatic pathways that induce apoptosis or other mechanisms of cell death (Choi, 1988;Tymianski et al., 1993;Ziv and Spira, 1995;Yoo et al., 2004). Cell death occurs more frequently for cells transected nearer to, compared to further from, their cell body (Ramón y Cajal, 1928) due to increased concentrations of somal Ca 2+ that probably disrupt somal protein synthesis (Yoo et al., 2004;Nguyen et al., 2005). ...
Transection of nerve axons (axotomy) leads to rapid (Wallerian) degeneration of the distal portion of the severed axon whereas the proximal portion and the soma often survive. Clinicians and neuroscientists have known for decades that somal survival is less likely for cells transected nearer to the soma, compared to further from the soma. Calcium ion (Ca2 +) influx at the cut axonal end increases somal Ca2 + concentration, which subsequently activates apoptosis and other pathways that lead to cell death. The same Ca2 + influx activates parallel pathways that seal the plasmalemma, reduce Ca2 + influx, and thereby enable the soma to survive. In this study, we have examined the ability of transected B104 axons to seal, as measured by uptake or exclusion of fluorescent dye, and quantified the relationship between sealing frequency and transection distance from the axon hillock. We report that sealing frequency is maximal about 150 μm (μm) from the axon hillock and decreases exponentially with decreasing transection distance with a space constant of about 40 μm. We also report that after Ca2 + influx is initiated, the curve of sealing frequency versus time is well-fit by a one-phase, rising exponential model having a time constant of several milliseconds that is longer nearer to, versus further from, the axon hillock. These results could account for the increased frequency of cell death for axotomies nearer to, versus farther from, the soma of many types of neurons.
... ATP production is reduced when the blood supply is insufficient, causing neuron polarization and accumulation of glutamate in the extracellular space due to reversed uptake (Rossi et al., 2000). This excess glutamate is responsible for neuron death in transient ischemia (Choi and Rothman, 1990), and its stimulation of massive calcium influx through the NMDA receptors plays a major role in 95 excitotoxicity (Tymianski et al., 1993a;Tymianski et al., 1993b where it may offer some protection against cell death. ...
Genome regulation is an extremely complex phenomenon. There are various mechanisms in place to ensure smooth performance of the organism. Post-transcriptional regulation of gene expression is one such mechanism. Many proteins bind to mRNAs and regulate their translation. In this thesis, I have focused on the Cytoplasmic Polyadenylation Element Binding family of proteins (CPEB1-4); a group of sequence specific RNA binding proteins important for cell cycle progression, senescence, neuronal function and plasticity. CPEB protein binds mRNAs containing a short Cytoplasmic Polyadenylation Element (CPE) in 3’ untranslated Region (UTR) and regulates the polyadenylation of these mRNAs and thereby controls translation. In Chapter II, I have presented my work on the regulation of mitochondrial function by CPEB. CPEB knockout mice have brain specific defects in mitochondrial function owing to a reduction in Electron transport chain complex I component protein NDUFV2. CPEB controls the translation of this NDUFV2 mRNA and thus affects mitochondrial function. A consequence of this reduced bioenergetics is reduced growth and branching of neurons, again emphasizing the importance of this pathway. Chapter III focuses on the role of CPEB4 in neuronal survival and protection against apoptosis. CPEB4 shuttles between nucleus and cytoplasm and becomes nuclear in response to stimulation with ionotropic glutamate receptors, focal ischemia in vivo and when cultured neurons are deprived of oxygen and glucose; nuclear CPEB4 affords protection against apoptosis in ischemia model. The underlying cause for nuclear translocation is reduction in Endoplasmic Reticulum calcium levels. These studies give an insight into the function and dynamics of these two RNA binding proteins and provide a better understanding of cellular biology.
... La neurotoxicidad del NMDAR (Sattler et al., 1999) viene determinada por la formación de canales con gran permeabilidad al Ca 2+ que se encuentran compuestos por subunidades NR1 y NR2 (Burnashev 1998 de ratones knock-out para la subunidad NR2A en los que la zona cerebral infartada tras la isquemia es menos extensa que en ratones no modificados genéticamente (Morikawa et al., 1998). Esta característica excitotóxica de la subunidad NR2A parece depender de su dominio C-terminal (Tymianski et al., 1993), ya que la eliminación de 400 aminoácidos en dicho extremo implica una reducción de la muerte celular (Anegawa et al., 2000). De hecho, en este dominio se producen las interacciones con muchas de las proteínas citoplásmicas que iniciarán la cascada isquémica (Kornau et al., 1995; Scannevin y Huganir 2000). ...
... The overwhelming theme of neuronal cell loss is that of an excitotoxic event by which prevention/correction cannot occur when the four neuroprotectors are not present in the aCSF and resulting from their concentrations being diminished during slice preparation. NMDAR mediated excitotoxicity is associated with an influx of Ca 2+ and Clions; Ca 2+ due to the activity of the receptor itself, which sets of a second messenger cascade resulting in necrotic or apoptotic cell death (Tymianski et al., 1993;Szydlowska and Tymianski, 2010) and Clto compensate for the excessive depolarisation excitotoxicity causes (Rothman, 1985;Aghajanian and Rasmussen, 1989;, Olney et al., 1986). It is this process that resulted in sucrose replacing NaCl in preparatory methods for acute brain slices (Aghajanian and Rasmussen, 1989). ...
Excitotoxic neuronal death, associated with neurodegenerative disorders and hypoxic insults, results from excessive exposure to excitatory neurotransmitters. Glutamate neurotoxicity is triggered primarily by massive Ca ²⁺ influx arising from overstimulation of the NMDA subtype of glutamate receptors. The underlying mechanisms, however, remain elusive. We have tested the hypothesis that mitochondria are primary targets in excitotoxicity by confocal imaging of intracellular Ca ²⁺ ([Ca ²⁺ ] i ) and mitochondrial membrane potential (ΔΨ) on cultured rat hippocampal neurons. Sustained activation of NMDA receptors (20 min) elicits reversible elevation of [Ca ²⁺ ] i . Longer activation (50 min) renders elevation of [Ca ²⁺ ] i irreversible (Ca ²⁺ overload). Susceptibility to NMDA-induced Ca ²⁺ overload is increased when the 20 min stimuli are applied to neurons pretreated with electron transport chain inhibitors, thereby implicating mitochondria in [Ca ²⁺ ] i homeostasis during excitotoxic challenges. Remarkably, ΔΨ exhibits prominent and persistent depolarization in response to NMDA, which closely parallels the incidence of neuronal death. Blockade of the mitochondrial permeability transition pore by cyclosporin A allows complete recovery of ΔΨ and prevents cell death. These results suggest that early mitochondrial damage plays a key role in induction of glutamate neurotoxicity.
The annexins are a family of calcium-dependent phospholipid-binding proteins. They are defined by the presence of a highly conserved 70 amino acid domain responsible for binding calcium ions and interacting with phospholipid membranes. All annexins contain four copies of this repeat except for annexin VI, which has eight. In addition, each annexin has a unique N-terminus, varying in size from around 10 to nearly 200 amino acids. Ten different annexins have been cloned in mammals and a further three in lower eukaryotic phyla. No functions have been unequivocally assigned to any members of the family. Most annexins have a restricted tissue distribution, but adl cells express at least one member of the family. A secretory cell line, rat basophilic leukaemia RBL-2H3 cells was shown to express annexins I, II, VI and VII, all of which have been implicated in exocytosis. Immunolabelling of annexin II in RBL-2H3 cells, revealed that it is localised to the cortical region and to punctate bodies in the cytosol. The presence of annexin II close to the plasma membrane, and possibly on secretory vesicles located in the cytosol, is consistent with a role in exocytosis. Several rat annexin II cDNA clones were isolated using a murine annexin II cDNA to screen a plasmid cDNA library. One of these was fully sequenced. Alignment of the rat annexin II cDNA sequence with those from other species revealed a 6-nucleotide insert in the coding region of one of the clones, close to the N-terminus. The insertion of 2 amino acids at this point may have important functional consequences since it is close to the sites of phosphorylation and the region that interacts with annexin II's cellular ligand, pll. To demonstrate the existence of this insert in vivo, primer extension analysis was performed on mRNA extracted from RBL-2H3 cells. To investigate the effect on secretion of a reduction in annexin II expression, RBL-2H3 cells were transfected with an expression plasmid containing rat annexin II cDNA in reverse orientation. A clone expressing significantly lower than wild type levels of annexin II was shown to have reduced secretory response to Ca2+ and GTPγS. Over a period of weeks this clone reverted to wild type levels of annexin II expression, and this coincided with a return to normal secretory response. The results in this thesis support the theory that annexin II functions in the secretory pathway.
Eukaryotic tissues are composed of individual cells surrounded by a plasmalemma that consists of a phospholipid bilayer with hydrophobic heads that bind cell water. Bound-water creates a thermodynamic barrier that impedes the fusion of a plasmalemma with other membrane-bound intracellular structures or with the plasmalemma of adjacent cells. Plasmalemmal damage consisting of small or large holes or complete transections of a cell or axon results in calcium influx at the lesion site. Calcium activates fusogenic pathways that have been phylogenetically conserved and that lower thermodynamic barriers for fusion of membrane-bound structures. Calcium influx also activates phylogenetically conserved sealing mechanisms that mobilize the gradual accumulation and fusion of vesicles/membrane-bound structures that seal the damaged membrane. These naturally occurring sealing mechanisms for different cells vary based on the type of lesion, the type of cell, the proximity of intracellular membranous structures to the lesion and the relation to adjacent cells. The reliability of different measures to assess plasmalemmal sealing need be carefully considered for each cell type. Polyethylene glycol (PEG) bypasses calcium and naturally occurring fusogenic pathways to artificially fuse adjacent cells (PEG-fusion) or artificially seal transected axons (PEG-sealing). PEG-fusion techniques can also be used to rapidly rejoin the closely apposed, open ends of severed axons. PEG-fused axons do not (Wallerian) degenerate and PEG-fused nerve allografts are not immune-rejected, and enable behavioral recoveries not observed for any other clinical treatment. A better understanding of natural and artificial mechanisms that induce membrane fusion should provide better clinical treatment for many disorders involving plasmalemmal damage.
Background:
Neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and cerebral ischemic stroke, impose enormous socio-economic burdens on both patients and health-care systems. However, drugs targeting these diseases remain unsatisfactory, and hence there is an urgent need for the development of novel and potent drug candidates.
Methods:
Animal toxins exhibit rich diversity in both proteins and peptides, which play vital roles in biomedical drug development. As a molecular tool, animal toxin peptides have not only helped clarify many critical physiological processes but also led to the discovery of novel drugs and clinical therapeutics.
Results:
Recently, toxin peptides identified from venomous animals, e.g. exenatide, ziconotide, Hi1a, and PcTx1 from spider venom, have been shown to block specific ion channels, alleviate inflammation, decrease protein aggregates, regulate glutamate and neurotransmitter levels, and increase neuroprotective factors.
Conclusion:
Thus, components of venom hold considerable capacity as drug candidates for the alleviation or reduction of neurodegeneration. This review highlights studies evaluating different animal toxins, especially peptides, as promising therapeutic tools for treatment of different neurodegenerative diseases and disorders.
The CA1 pyramidal neurons in the hippocampus are selectively vulnerable to transient ischemic damage. In experimental animals, the CA1 pyramidal neurons undergo cell death several days after brief forebrain ischemia. It remains, however, unknown whether this delayed neuronal death is necrosis or apoptosis. To investigate the degenerating processes of the CA1 pyramidal neurons in gerbil hippocampus after brief ischemia, lysosomal and nuclear alterations in the cells were examined using immunocytochemistry, in situ nick-end labeling, and Southern blotting. By light and electron microscopy, immunoreactivity for cathepsins B, H, and L, representative lysosomal cysteine proteinases, increased in the CA1 pyramidal neurons 3 d after ischemic insult, which showed cell shrinkage. By morphometric analysis, the volume density of cathepsin B-positive lysosomes markedly increased 3 d after ischemic insult, while that of autophagic vacuole-like structures also increased at this stage, suggesting that cathepsin B-immunopositive lysosomes increasing in the neurons after ischemic insult are mostly autolysosomes. Nuclei of the CA1 neurons were nick-end labeled by biotinylated dUTP mediated by terminal deoxytransferase 3 and 4 d after ischemic insult, but not in the prior stages. Simultaneously, dense chromatin masses appeared in nuclei of the neurons. By Southern blotting, laddering of DNA occurred only in CA1 hippocampal tissues obtained 4 d after ischemic insult. Confocal laser scanning microscopy demonstrated that the fragmented DNA in the CA1 pyramidal layer was phagocytosed by microglial cells. The results suggest that delayed death of the CA1 pyramidal neurons after brief ischemia is not necrotic but apoptotic.
Glutamate (Glu) is the major excitatory neurotransmitter in the mammalian central nervous system (CNS). Excessive glutamate in the extracellular space can trigger passive and active forms of neuronal death in the CNS via excessive activation of glutamate receptors. This phenomenon has been named excitotoxicity. Excitotoxicity has been implicated in the pathogenesis of acute and chronic neurodegenerative disorders. However, despite overwhelming preclinical data, human clinical trials in stroke or traumatic brain injury with antiexcitotoxic compounds acting at different levels of the excitotoxic cascade have failed to provide the expected neuroprotective effect. Here we present a focused overview of excitotoxic research including physiology of glutamate receptors and intracellular pathways leading to cell death. We review evidence for involvement of excitotoxicity in human neurodegenerative disorders and the results of several clinical trials. We present the most recent trends in the development of antiexcitotoxic therapies and also briefly refer to the hypothesis that interference with the trophic actions of glutamate in the context of CNS injuries may be one of the reasons why clinical trials with antiexcitotoxic compounds have failed. © Springer Science+Business Media New York 2014. All rights reserved.
Over the past several decades, an overwhelming body of research has greatly expanded our understanding of the mechanisms underlying excitotoxicity in brain ischemia as well as in many chronic neurodegenerative diseases. The identification of an array of molecular targets has opened avenues for neuroprotective strategies and, consequently, has sparked considerable interest for their attractive therapeutic means as pharmacological options. The purpose of this work is to provide a general overview of neuronal excitotoxicity and the inevitable downstream consequences of Ca2+ overload. We also discuss the contribution of Ca2+ transporters in excitotoxicity. This article is part of a Special Issue entitled "Calcium Pumps and Exchangers in Neuronal Injury and Neurodegeneration".
Activation of extrasynaptic NMDA receptors by high glutamate concentrations is one of the key pathogenic factors following a stroke. For this reason, the search for efficient neuroprotective agents that could reduce glutamate toxicity is a pressing need. Ca²⁺ overload in response to glutamate leads to activation of signaling cascades in the cell and the development of oxidative stress, which ultimately leads to apoptosis. Using a model system of acute excitotoxicity caused by 50 μM NMDA, which was used as a specific NMDA receptor activator, we demonstrated that during 2 hours of incubation the viability of the primary neuronal culture decreased by 30–50%. To demonstrate that the observed effect is associated not only with the Ca²⁺ influx into the cytoplasm through the activated NMDA receptors, we decreased the Ca²⁺ concentration in the medium. The lowered Ca2+ concentration, as well as its complete absence, did not affect NMDA toxicity. We tested carnosine, a naturally occurring dipeptide and promising antioxidant, as a neuroprotective agent. The addition of 2 mM carnosine prevented the decrease in cell viability caused by a 2-hour incubation with 50 μM NMDA, while it showed no effect on the viability of the cell culture in the control. Based on the results, we consider the further study of carnosine, its complexes, and analogues as neuroprotectors in cerebral ischemia promising.
Spinal cord ischemia reperfusion injury (SCIRI) can cause spinal cord dysfunction and even devastating paraplegia. Calcium-sensing receptor (CaSR) and calpain are two calcium related molecules which have been reported to be involved in the ischemia reperfusion injury of cardiomyocytes and the subsequent apoptosis. Here, we studied the expression of CaSR and calpain in spinal cord neurons and tissues, followed by the further investigation of the role of CaSR/calpain axis in the cellular apoptosis process during SCIRI. The results of in vitro and in vivo studies showed that the expression of CaSR and calpain in spinal cord neurons increased during SCIRI. Moreover, the CaSR agonist GdCl3 and antagonist NPS-2390 enhanced or decreased the expression of CaSR and calpain respectively. The expressions of CaSR and calpain were also consistent with the cellular apoptosis in spinal cord. Taken together, CaSR-calpain contributes to the SCIRI apoptosis, and CaSR antagonist might be a helpful drug for alleviating SCIRI.
We compared how different metabolic stressors, anoxic coma and food deprivation, affected signaling in neural tissue. We used the locust's Descending Contralateral Movement Detector (DCMD) interneuron because its large axon, high firing frequencies, and rapid conduction velocity make it energetically expensive. We exposed locusts to a 30 minute anoxic coma or 1 day of food deprivation and found contrasting effects on signaling within the axon. After a prior anoxic coma, the DCMD fired fewer high-frequency (> 200 Hz) action potentials (APs) (Control: 12.4 ± 1.6; Coma: 6.3 ± 0.9) with a reduction in axonal conduction velocity (CV) at all frequencies (∼4-8%) when presented with a standard looming visual stimulus. Prior anoxic coma was also associated with a loss of supernormal conduction by reducing both the number of supernormal APs and the firing frequency with the highest CV. Initially, food deprivation caused a significant increase in the number of low- and high-frequency APs with no differences observed in CV. After controlling for isolation, food deprivation resulted in an increase in high-frequency APs (>200Hz: Control: 17.1 ± 1.7; Food-deprived: 19.9 ± 1.3) and an increase in relative conduction velocity for frequencies >150Hz (∼2%). Action potentials of food-deprived animals had a smaller half-width (Control: 0.45 ± 0.02ms; Food-deprived: 0.40 ± 0.01ms) and decay time (Control: 0.62 ± 0.03ms; Food-deprived: 0.54 ± 0.02ms). Our data indicate that the effects of metabolic stress on neural signaling can be stressor-dependent.
Substantial efforts are being made by pharmaceutical industries to develop drugs which will protect the brain from neurodegeneration that follows a variety of diseases. Stroke is the third leading cause of mortality and a major cause of long-lasting physical disability. A key factor in recent drug discovery efforts was the development of animal tests that mimic the neuropathological consequences of stroke by occlusion of the middle cerebral artery in rats, dogs and primates. Beside causing substantial stress, pain and anxiety to the animals these in vivo bioassays suffer from several problems mostly arising from their inter- and intra-assay variability. However, major advances have recently been made in our understanding of the underlying pathocellular mechanisms associated with brain injury. The demonstration, that disruption in calcium homeostasis initiated in part by excessive excitatory amino acid (EAA) release and overstimulation of EAA receptors mediates neuronal injury, has opened up new cytotherapeutic avenues, such as EAA receptor antagonists that would be susceptible to interrupt the cascade of cellular events leading to excitotoxic cell death.
These findings led us to the assumption that monitoring of calcium deregulation in individual cells could also be used in drug screening as a predictive marker of neuronal injury. The objective of our ongoing studies therefore is to develop an in vitro assay in which individual neurons can be quantitatively studied under neurotoxic conditions with respect to their calcium dynamics and the temporal relationship between intracellular calcium accumulation and cell death. Using digitized fluorescence imaging we demonstrate the value of this non-invasive technique to continuously observe the dynamic intracellular calcium concentration and cell death in parallel in order to evaluate the neuroprotective or neurotoxic effect of a test compound.
Excitotoxicity refers to neuronal death caused by activation of excitatory amino acid receptors. Several lines of evidence have linked excitotoxic cell death to the pathogenesis of both acute and chronic neurologic diseases. The initial observation that glutamate was neurotoxic was that of Lucas and Newhouse, who found that administration of glutamate to mice resulted in retinal degeneration (Lucas and Newhouse, 1957). Subsequent studies of Olney and colleagues linked neurotoxicity to the activation of excitatory amino acid receptors, and the term “excitotoxin” was coined (Olney, 1969). Further advances were those of Rothman linking release of excitatory amino acids to anoxic cell death in hippocampal cultures (Rothman, 1984), and of Choi linking calcium influx to delayed cell death caused by excitatory amino acids (Choi, 1987). More work has linked activation of excitatory amino acid receptors to free radical generation and nitric oxide, both of which may lead to oxidative stress (Dawson et al., 1991: Lafon-Cazal et al., 1993).
Calcium ions (Ca2+) are ubiquitous intracellular second messengers which regulate numerous cellular functions. For this reason, neurons possess a complex homeostatic machinery which tightly controls the temporal and spatial distribution of Ca2+ within the cell. It is widely believed that a disruption of Ca2+ homeostasis is a major contributing factor to neuronal cell death occurring in many of the nervous system disorders including cerebral hypoxia/ischemia, brain trauma, inflammatory, and degenerative diseases (also see chapters by Leist and Nicotera, Dykens, Olney and Ishimaru, Dietrich, Wood, Vornov, Shin and Lee, and Bar-Peled and Rothstein). Therefore, many therapeutic strategies for these disorders aim at preventing either the processes leading to Ca2+ homeostatic failure, or the consequences of Ca2+ excess. Recent insights into mechanisms triggering and perpetuating disturbances of cellular calcium regulation have led to new approaches to the study of cellular calcium homeostasis, and to treatment of neuronal injury. One such approach, which illustrates the complexities associated with Ca2+ regulation, has been the use of exogenous Ca2+- chelating agents. These compounds have been employed in recent years to study normal and pathological cellular Ca2+ signaling, to evaluate the role of Ca2+ buffering in neuronal vulnerability to injury, and to treat neuronal injuries thought to be associated with Ca2+ homeostatic failure. The purpose of this chapter, therefore, is to examine current knowledge on the role of Ca2+ buffers as probes of mechanisms leading to nerve cell death, and as potential therapeutic agents for Ca2+-dependent neurotoxicity.
It is now recognized that neural death associated with acute insults such as trauma, hypoglycemia, seizures, global hypoxia, and stroke is due in part to calcium-mediated excitotoxic injury (for review see ref. [1]). More specifically, acute insult stimulates excessive release of excitatory amino acid neurotransmitters (EAAs) that in turn cause a rapid influx of calcium ions in affected neurons (2). Intracellular calcium is essential for normal neural function; however, calcium overload may disrupt cell metabolism, cell excitability, gene expression, and other vital functions (3). Although the process by which calcium-mediated excitotoxic injury occurs may vary (4), the end result of compromised cell function inevitably contributes to cell death.
Glutamate is believed to be the key mediator in neurodegenerative processes associated with stroke, epilepsy, and a broad spectrum of neurodegenerative disorders, including, Huntington’ s disease and Alzheimer disease (Choi, 1988; Beal, 1993). The elucidation of the underlying mechanism involved in the pathophysiology of glutamate has been the topic of intensive research. Although the role of glutamate-induced neurotoxicity in chronic neurodegenerative disease remains unclear, compelling evidence suggests that ischemia-triggered neuronal damage is largely attributed to excessive and persistent activation of glutamate receptors (for recent reviews, see Whetsell and Shapira, 1993; Choi, 1988; Beal, 1933). Because glutamate can produce both excitation and toxicity in neurons, the term “excitotoxicity” was proposed by Olney and coworkers (Olney et al., 1978). It is now widely believed that excessive elevation of intracellular calcium is an early and critical step in excitotoxicity (see Fig 1; and Rothman, 1984; Choi, 1988; Garthwaite and Garthwaite, 1986; Frandsen and Schousboe, 1993). In vivo evidence supporting a central role of calcium in excitotoxicity is derived from the observation that calcium accumulates in nervous tissue in cerebral ischemia (Siesjo and Bengtsson, 1989; Simon et al., 1984) and in epilepsy (Meyer, 1989; Uematsu et al., 1990), and that ischemic cell damage can be attenuated by suppression of calcium influx (Pizzi et al., 1991; Valentino et al., 1993) and chelation of intracellular calcium (Tymianski et al., 1993).
Organogenesis requires the death of specific cells at genetically determined time points during development. This type of cell death, known as programmed cell death or apoptosis, plays a major role in the normal development of most organs including the immune and central nervous systems. Kerr and coworkers first described the ultrastructural changes characteristic of dying cells (Kerr et al., 1972) and since then few fields have attracted as much attention as apoptosis. Apoptosis is a natural process, and its dysregulation is pathologic. For example, apoptosis is believed to contribute to neuronal loss during stroke or central nervous system trauma and in neurodegenerative disorders including Alzheimer’s and Parkinson’s disease. Conversely, lack of apoptosis is known to result in tumorigenesis. Cells are genetically programmed for self-repair following pathological insult. Under normal conditions when self-repair is unsuccessful a series of highly regulated suicide signaling pathways are initiated. Apoptosis is triggered by extrinsic or intrinsic inducers that activate pathways leading to DNA cleavage. Emerging paradigms for apoptotic signaling include: 1) the requirement for translocation of proteins from the cytosol to the nucleus; and 2) calcium (Ca2+)-dependent activation of phosphatases required for this translocation. Cytosolic Ca2+ is a second messenger that regulates signaling required for cellular activation, proliferation, differentiation and cell death. Cytosolic Ca2+ is rigidly maintained at ~100 nM in resting cells and its elevation to levels between 500 and 1000 nM activates numerous signaling pathways in all types of cells. Levels above 1000 nM are generally toxic. Ca2+ channels in the plasma membrane (both voltage- and ligand-gated), and ligand-gated Ca2+-release channels in the endoplasmic reticulum are the most important sources of cytosolic Ca2+. Conversely, Ca2+-ATPases on endoplasmic reticulum and the plasma membrane are the most important pathways for removal of cytosolic Ca2+.
Calcium (Ca2+) ions are ubiquitous intracellular messengers governing innumerable functions such as the control of cell growth and differentiation, membrane excitability, exocytosis and synaptic activity. Because of this, neurons must tightly regulate the cytosolic Ca2+ concentration ([Ca2+]i) to achieve a sufficiently high signal-to-noise ratio for efficient Ca- signaling to occur. The resting free [Ca2+]i must remain at very low levels (around 100 nM, or 105 times lower than extracellular [Ca2+]), so that relatively small or localized increases in [Ca2+]i can be used to trigger physiological events such as the activation of an enzyme or an ion channel. Neurons have therefore evolved complex homeostatic mechanisms to control both [Ca2+]i and the intracellular location of Ca2+ ions (for a general review of Ca2+ homeostasis in neurons see refs Blaustein, 1988; Meldolesi et al. 1988; Smith, Augustine, 1988; Miller, 1992). These mechanisms consist of complex interactions between four general categories of events: Ca2+ influx, Ca2+ buffering, internal Ca2+ storage and Ca2+ efflux. Under physiological conditions, a delicate interplay between these processes allows multiple Ca2+ dependent signaling cascades to be regulated independently within the same cell. However, it is widely believed that excessive Ca2+ loading, exceeding the capacity of Ca-regulatory mechanisms, may inappropriately activate Ca-dependent processes which either lie dormant or normally operate at low levels. When over-activated, such processes directly damage neurons or lead to the formation of toxic reaction products which ultimately cause cell death. However, in spite of two decades of research supporting the association between Ca2+ excess and neurotoxicity, the precise molecular mechanisms by which Ca2+ toxicity occurs remain poorly understood.
Disruption of cellular regulation of calcium has long been implicated in the mechanism of neruonal death following cerebral ischemia and reperfusion9,17. While much research has been directed at the potential role of calcium influx in ischemia, less attention has been paid to other mechanisms of calcium regulation that may be altered. We are investigating the possibility that altered storage and release of Ca2+ from the endoplasmic reticulum (ER) contributes to ischemic neuronal death.
The element oxygen (O) exists in air as a molecule (O2) known as dioxygen or molecular oxygen. It was first isolated and characterized between 1772–1774 by the individual skills of the great European scientists Lavoisier and Scheele. Dioxygen, hereafter referred to as oxygen, appeared in significant amounts on the surface of the Earth some 2.5 × 109 years ago, and geological evidence suggests that this was due to the photosynthetic activity of micro-organisms (blue-green algae). The slow and steady rise in atmospheric oxygen concentration was accompanied by the formation of the ozone layer in the stratosphere. Both oxygen and the ozone layer acted as critical filters against the intense solar ultraviolet light reaching the surface of the Earth. The universe exists predominantly as hydrogen (H) and helium (He) with the Earth as a unique center of oxidation in an otherwise reducing universe.
The premise of this chapter is that disruption of cellular calcium homeostasis plays a pivotal role in the neurodegenerative process that occurs in many different neurological disorders, with a focus on cerebral ischemia. I will not attempt to review the extensive literature supporting the involvement of calcium in ischemic injury to neurons (see Choi 1995; Mattson and Mark 1996 for review), but rather will provide selected examples of such evidence, together with a more thorough account of preventative and therapeutic approaches that target systems involved in either disruption or stabilization of neuronal calcium homeostasis. A variety of cell culture and animal models of neurodegenerative processes relevant to ischemic brain injury have been developed. Such experimental models have allowed the manipulation and monitoring of Ca2+ movements in living neurons exposed to conditions relevant to humans suffering from stroke and other neurodegenerative conditions, thus clarifying roles of calcium in neuronal cell death processes. Other chapters in this volume expand upon some of the mechanisms that contribute to the sustained elevations of intracellular free calcium level ([Ca2+]i) that occur in neurons following ischemic or traumatic insults — these include energy failure, overstimulation of glutamate receptors, and oxidative stress.
All neurons in the central nervous system of mammals express receptors for the excitatory amino acid glutamate. Although glutamatergic neurotransmission is therefore essential for the functioning of neuronal circuits in the brain and spinal cord, under certain conditions activation of glutamate receptors can trigger the death of neurons. Such excitotoxicity most often occurs when cells are coincidentally subjected to reduced levels of oxygen or glucose, increased levels of oxidative stress, trauma, or exposure to toxins or other pathogenic agents. Excitotoxicity is mediated by excessive calcium influx and release from internal organelles, oxyradical production and the activation of a form of programmed cell death called apoptosis. Proteins such as p53, Bax and Par-4 induce mitochondrial membrane permeability changes resulting in the release of cytochrome c and the activation of proteases such as caspase-3. Essentially all subcellular compartments, including the endoplasmic reticulum, mitochondria and nucleus are involved in the excitotoxic process. Excitotoxic cascades are initiated in postsynaptic dendrites where glutamate receptors are most highly concentrated, and may either cause local degeneration or plasticity of those synapses or may propagate the signals to the cell body resulting in cell death. The nervous system protects itself against excitotoxicity by deploying multiple antiexcitotoxic signaling pathways including neurotrophic signaling pathways, intrinsic stress-response pathways, and survival proteins such as protein chaperones, calcium-binding proteins and inhibitor of apoptosis proteins. A rapid accumulation of information on the molecular underpinnings of the excitotoxic process is leading to the development of novel therapeutic approaches for neurodegenerative disorders, as well as unexpected insight into mechanisms of synaptic plasticity.
This chapter describes the role of calcium ions in necrotic and apoptotic cell death. The role of calcium ion as intracellular regulator of many physiological processes is well established. The alterations in Ca2+ signaling can also affect cell functions and play a determinant role in a variety of pathological and toxicological processes. Studies using selective indicators have shown that the Ca2+ concentration in the cytosol of unstimulated cells is maintained between 0.05 and 0.2 μM. Slow propagating Ca2+ waves have been detected after agonist stimulation in some cell types, suggesting that Ca2+ fluctuations are not uniform in time and spatial distribution. The findings that removal of extracellular Ca2+, or pretreatment with intracellular Ca2+ chelators, can prevent both the nuclear changes typical of apoptosis and cell lysis, have provided support for the idea that Ca2+ signals can initiate apoptosis in some systems. In many in vitro models of apoptosis the loss of intracellular Ca2+ homeostasis is accompanied by the activation of a Ca2+-dependent endonuclease activity. Intracellular Ca2+ overload due to excessive stimulation of excitatory amino acid receptors and enhanced Ca2+ influx through membrane channels appears to play an important role in ischaemic brain damage.
Stroke remains one of the major causes of death and disability throughout the world (American Heart Association, 1991). More than 80% of all strokes are a result of cerebral ischemia (Mohr et al., 1978). Global cerebral ischemia involves the entire brain and occurs during cardiac arrest or severe systemic hypotension. Focal cerebral ischemia affects restricted brain regions and occurs in a wide variety of clinical settings but is most commonly a result of cerebral vascular atherosclerosis. Focal ischemia is more frequent than global ischemia.
The major excitatory transmitter in the mammalian central nervous system is glutamate, which exerts its signaling actions through the stimulation of ionotropic and metabotropic receptors (Watkins et al. 1981; Mayer and Westbrook 1987; Nakanishi and Masu 1994). Under pathological conditions, glutamate receptor overactivation can trigger neuronal death, a phenomenon known as excitotoxicity (Lucas and Newhouse 1957; Olney 1969). Incentive for developing practical methods for blocking excitotoxicity arises from its implication in several acute and chronic neurological disease states. While recent clinical trials aimed at blocking excitotoxicity in stroke patients have been disappointing, there are several plausible reasons for these trial failures, including specific study design issues, treatment side effects, and a need to achieve concurrent block of parallel injury pathways. In our view, the case for antiexcitotoxic approaches in stroke remains open, and there are other possible disease targets yet to be explored. Ongoing delineation of the cellular and molecular underpinnings of excitotoxicity has led to the progressive unveiling of countermeasures, aimed at attenuating presynaptic glutamate release, postsynaptic receptor activation, the movement or action of cation second messengers, or downstream intracellular injury cascades. The excitotoxicity concept itself may need to be expanded, to encompass the death of oligodendrocytes as well as neurons, and ionic derangements besides Ca2+ overload.
The key role of Ca2+ ions (Ca2+) for the function of excitable tissues was already described in 1882 in the classical experiments by Ringer. Since then, a plethora of cellular functions have been found to require Ca2+ signals or simply the maintenance of a set Ca2+ concentration. The major requirement for the signaling function of Ca2+ is the existence of a concentration gradient between the interstitial fluid and the interior of the cell. Whereas extracellular concentrations of free Ca2+ range between 1.3 and 1.8 mM, the free intracellular Ca2+ concentration ([Ca2+]i) is about 100 nM. Thus, cells are continuously faced with the problem of maintaining a concentration gradient of more than four orders of magnitude across the plasma membrane. Various physiological stimuli increase [Ca2+]i transiently and thereby induce cellular responses. However, under pathological conditions, changes of [Ca2+]i are generally more pronounced and sustained. Pronounced elevations of [Ca2+]i activate hydrolytic enzymes, lead to exaggerated energy expenditure, impair energy production, initiate cytoskeletal degradation, and ultimately result in cell death. Such Ca2+-induced cytotoxicity may play a major role in several neuropathological phenomena including chronic neurodegenerative diseases as well as acute neuronal losses (i.e., during the pathogenesis of stroke).
Excitotoxicity refers to neuronal death caused by the overactivation of excitatory amino acid receptors. Several lines of evidence have linked excitotoxicity to the pathogenesis of both acute and chronic neurologic diseases. Research into the mechanisms of excitotoxic injury has associated activation of excitatory amino acid receptors to free radical generation and nitric oxide, which in turn leads to oxidative stress. Downstream enzymatic effectors include a mix of proteases, free radicals, and endonucleases. Excitatory amino acids play a role in acute neurological diseases such as stroke, trauma, and hypoglycemia; their role in chronic neurological diseases too has been supported by studies using animal models and by work with a glutamate release inhibitor in amyotrophic lateral sclerosis. Chronic diseases linked to excitotoxicity include Alzheimer's disease, Huntington's disease, and Parkinson's disease. Despite the overwhelming evidence of excitotoxicity in these acute and chronic diseases and a variety of pharmacological interventions aimed at inhibiting these processes, very few treatments have shown efficacy in clinical trials. As such, recent research has been directed at nonglutamate ion channels responsible for ionic imbalance as well as cross-talk between cell death pathways implicated in excitotoxicity.
Introduction, In 1935 Krebs discovered that the amino acid glutamate increases metabolism in the isolated retina and that it is concentrated in the cerebral gray matter (Krebs, 1935). Hayashi (1952, 1958) first reported on excitatory properties of glutamate on neuronal tissue. Local administration of glutamate on the motor cortex of dogs and primates resulted in motor seizures. Curtis et al. (1959) subsequently demonstrated that glutamate and aspartate, when applied iontophoretically to the cat spinal cord, depolarized neurons. Since the 1960s there has been appreciation of the role of glutamate in the nervous system, and today it is considered the major excitatory neurotransmitter (Fonnum, 1984). It is essential for learning and memory, synaptic plasticity, neuronal survival and, in early development, for proliferation, migration and differentiation of neuronal progenitors and immature neurons (Guerrini et al., 1995; Ikonomidou et al., 1999; Komuro & Rakic, 1993). Glutamate fulfils its various functions due to its compartmentalization (Fonnum, 1984). The largest pool of glutamate is the metabolic pool. The neuronal pool is located in nerve endings and represents the neurotransmission pool. A separate pool is located in glia and serves the recycling of transmitter glutamate. The smallest glutamate pool is involved in synthesis of the inhibitory neurotransmitter γ-aminobutyric acid (GABA). Glutamate is released from presynaptic terminals by a calcium-dependent mechanism, is removed subsequently by uptake into the surrounding glial cells and aminated to glutamine.When released into the synaptic cleft, glutamate acts at the postsynaptic site on receptors (Hollmann & Heinemann, 1994; Nakanishi, 1992).
Increasing evidence has implicated oxidative damage in the pathogenesis of neurodegenerative diseases. The major source of free radicals in the cell is the mitochondria. Peroxynitrite is formed by the reaction of superoxide with nitric oxide, and it produces both oxidative damage and protein nitration. Mutations in CuZn superoxide dismutase associated with familial ALS may result in increased −OH radical generation or in increased reactivity with peroxynitrite to nitrate proteins. There is evidence for increased oxidative damage in Alzheimer's disease and Parkinson's disease in neurons undergoing neurodegenerative changes. A role for oxidative damage in Parkinson's disease toxicity and in Huntington's disease is supported by studies in animal models. Improved antioxidant therapies may prove useful in slowing or halting the progression of neurodegenerative diseases.
The end of the dispute between L.M. Ericsson and Qualcomm Corp. on the intellectual property rights to code division multiple access (CDMA) technology for 3G wireless air-interface has cleared the way towards the development of standards for third generation (3G) digital communication systems. These systems include packet- and switched-circuit based mobile communications with a digital bandwidth up to 2 Mbit/s, which will be offered at frequencies in the radio spectrum around 2 GHz in the near future. The settlement agreement binds the two companies to support a CDMA radio interface standard that has multiple modes of operation allowing different radio interfaces to be used in different parts of the world.
A new family of highly fluorescent indicators has been synthesized for biochemical studies of the physiological role of cytosolic free Ca2+. The compounds combine an 8-coordinate tetracarboxylate chelating site with stilbene chromophores. Incorporation of the ethylenic linkage of the stilbene into a heterocyclic ring enhances the quantum efficiency and photochemical stability of the fluorophore. Compared to their widely used predecessor, “quin2”, the new dyes offer up to 30-fold brighter fluorescence, major changes in wavelength not just intensity upon Ca2+ binding, slightly lower affinities for Ca2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca2+, should make these dyes the preferred fluorescent indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.
The cellular mechanisms by which excess exposure to the excitatory neurotransmitter glutamate can produce neuronal injury are unknown. More than a decade ago it was hypothesized that glutamate neurotoxicity (GNT) is a direct consequence of excessive neuronal excitation (“excitotoxicity” hypothesis); more recently, it has been hypothesized that a Ca influx triggered by glutamate exposure might mediate GNT (Ca hypothesis). A basic test to discriminate between these hypotheses would be to determine the dependence of GNT on the extracellular ionic environment. The excitotoxicity hypothesis predicts that GNT should depend critically on the presence of extracellular Na, since that ion appears to mediate glutamate neuroexcitation in the CNS; the Ca hypothesis predicts that GNT should depend critically on the presence of extracellular Ca. The focus of the present experiments was to determine the effects of several alterations in the extracellular ionic environment upon the serial morphologic changes that occur after mouse neocortical neurons in cell culture receive toxic exposure to glutamate. The results suggest that GNT in cortical neurons can be separated into 2 components distinguishable on the basis of differences in time course and ionic dependence. The first component, marked by neuronal swelling, occurs early, is dependent on extracellular Na and Cl, can be mimicked by high K, and is thus possibly “excitotoxic.” The second component, marked by gradual neuronal disintegration, occurs late, is dependent on extracellular Ca, can be mimicked by A23187, and is thus possibly mediated by a transmembrane influx of Ca. While either component alone is ultimately capable of producing irreversible neuronal injury, the Ca-dependent mechanism predominates at lower exposures to glutamate. Glutamate exposure likely leads to a Ca influx both through glutamate-activated cation channels and through voltage- dependent Ca channels activated by membrane depolarization. Addition of 20 mM Mg, however, did not substantially block GNT; this finding, together with the observation that GNT is largely preserved in sodium- free solution, supports the notion that the activation of voltage- dependent Ca channels may not be required for lethal Ca entry. The possibility that N-methyl-D-aspartate receptors may play a dominant role in mediating glutamate-induced lethal Ca influx is discussed.
Glutamatergic transmission is an important factor in the development of neuronal death following transient cerebral ischemia. In this investigation the effects of N-methyl-D-aspartate (NMDA) and non-NMDA receptor antagonists on neuronal damage were studied in rats exposed to 10 min of transient cerebral ischemia induced by bilateral common carotid occlusion combined with hypotension. The animals were treated with a blocker of the ionotropic quisqualate or alpha-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) receptor, 2.3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX), given postischemia as an intraperitoneal bolus dose of 30 mg kg-1 followed by an intravenous infusion of 75 micrograms min-1 for 6 h, or with the noncompetitive NMDA receptor blocker dizocilpine (MK-801) given 1 mg kg-1 i.p. at recirculation and 3 h postischemia, or with the competitive NMDA receptor antagonist DL-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid (CGP 40116), 5 mg kg-1, given intraperitoneally at recirculation. Treatment with NBQX provided a significant reduction of neuronal damage in the hippocampal CA1 area by 44-69%, with the largest relative decrease in the temporal part of the hippocampus. In neocortex a significant decrease in the number of necrotic neurons was also noted. No protection could be seen following postischemic treatment with dizocilpine or CGP 40116. Our data demonstrate that AMPA but not NMDA receptor antagonists decrease neuronal damage following transient severe cerebral ischemia in the rat and that the protection by NBQX may be dependent on the severity of the ischemic insult. We propose that the AMPA receptor-mediated neurotoxicity could be due to ischemia-induced changes in the control mechanisms of AMPA receptor-coupled processes or to changes of AMPA receptor characteristics.
Increases in intracellular calcium concentration are required for the release of neurotransmitter from presynaptic terminals
in all neurons. However, the mechanism by which calcium exerts its effect is not known. A low-sensitivity calcium-dependent
photoprotein (n-aequorin-J) was injected into the presynaptic terminal of the giant squid synapse to selectively detect high
calcium concentration microdomains. During transmitter release, light emission occurred at specific points or quantum emission
domains that remained in the same place during protracted stimulation. Intracellular calcium concentration microdomains on
the order of 200 to 300 micromolar occur against the cytoplasmic surface of the plasmalemma during transmitter secretion,
supporting the view that the synaptic vesicular fusion responsible for transmitter release is triggered by the activation
of a low-affinity calcium-binding site at the active zone.
Glutamate-induced changes in intracellular free Ca2+ concentration ([Ca2+]i) were recorded in single rat hippocampal neurons grown in primary culture by employing the Ca2+ indicator indo-1 and a dual-emission microfluorimeter. The [Ca2+]i was monitored in neurons exposed to 100 microM glutamate for 5 min and for an ensuing 3 hr period. Ninety-two percent (n = 64) of these neurons buffered the glutamate-induced Ca2+ load back to basal levels after removal of the agonist; thus, the majority of cells had not lost the ability to regulate [Ca2+]i at this time. However, following a variable delay, in 44% (n = 26) of the neurons that buffered glutamate-induced Ca2+ loads to basal levels, [Ca2+]i rose again to a sustained plateau and failed to recover. The changes in [Ca2+]i that occur during glutamate-induced delayed neuronal death can be divided into three phases: (1) a triggering phase during which the neuron is exposed to glutamate and the [Ca2+]i increases to micromolar levels, followed by (2) a latent phase during which the [Ca2+]i recovers to a basal level, and (3) a final phase that begins with a gradual rise in the [Ca2+]i that reaches a sustained plateau from which the neuron does not recover. This delayed Ca2+ overload phase correlated significantly with cell death. The same sequence of events was also observed in recordings from neuronal processes. The delayed Ca2+ increase and subsequent death were dependent upon the presence of extracellular Ca2+ during glutamate exposure. Calcium influx during the triggering phase resulted from the activation of both NMDA and non-NMDA receptors as indicated by studies using receptor antagonists and ion substitution. Treatment with TTX (1 microM) or removal of extracellular Ca2+ for a 30 min window following agonist exposure failed to prevent the delayed Ca2+ overload. The delayed [Ca2+]i increase could be reversed by removing extracellular Ca2+, indicating that it resulted from Ca2+ influx. The three phases defined by changes in the [Ca2+]i during glutamate-induced neuronal toxicity suggest three distinct targets to which neuroprotective agents may be directed.
The regulation of synaptic transmission by Ca(2+)-activated potassium (gKca) channels was investigated at the frog neuromuscular junction (nmj). Charybdotoxin (CTX), a blocker of certain types of gKca channels, induced a twofold increase of transmitter release. Similar results were obtained with purified natural toxin, synthetic toxin, and recombinant toxin. Apamin, a blocker of a different type of gKca channel, did not alter transmitter release. CTX was ineffective after intraterminal Ca2+ buffering was increased by application of the membrane-permeant Ca2+ buffer dimethyl-BAPTA-AM. By itself, the permeant buffer first caused a slight increase in transmitter release before release was eventually decreased. This increase of release did not occur when the buffer was applied in the presence of CTX or Ba2+, another gKca channel blocker. Stimulus-evoked entry of Ca2+ in nerve terminals, detected with the fluorescent Ca2+ indicator FLUO-3, was increased after blockade of gKca channels by CTX. CTX had no effect on the amount or the time course of synaptic depression. The results are consistent with the hypothesis that CTX-sensitive gKca channels normally narrow the presynaptic action potential and thus, by indirectly regulating Ca2+ entry, can serve as powerful modulators of evoked transmitter release. In order to affect presynaptic action potentials, the gKca channels must be located close to Ca2+ channels.
1. The postnatal development of membrane properties and outward K+ currents in CA1 neurons in rat hippocampal slices was studied with the use of whole-cell patch-clamp techniques. 2. Neurons at all postnatal ages (2-30 days; P2-30) were capable of generating tetrodotoxin (TTX)-sensitive action potentials in response to intracellular injection of depolarizing current pulses. There was a gradual increase in the amplitude and a decrease in the duration of these action potentials with age. Stable values for spike duration were reached by P15, whereas spike amplitude increased until P20-25. In P2-5 neurons, the duration of action potentials was greatly prolonged by depolarization from the resting membrane potential, indicating a weak spike repolarizing mechanism at depolarized potentials. In contrast, the duration of spikes evoked in P20-30 neurons was not affected by similar changes in the membrane potential. 3. Application of tetraethylammonium (TEA, 10 mM) had no effect on the duration of spikes in P3-5 neurons, whereas application of 4-aminopyridine (4-AP, 2 mM) produced large increases in spike duration. In contrast, the duration of spikes in P26 neurons was greatly increased after TEA application, whereas 4-AP had smaller effects on spike duration in these neurons. 4. The input resistance and membrane time constant decreased with age from P2 to P15. The values for both parameters were considerably greater than those reported with conventional intracellular recording electrodes in the immature hippocampus. The resting membrane potential became more hyperpolarized with age. When the recording pipettes contained KCl (140 mM), the resting potential of P3-4 neurons was 34 mV depolarized compared with resting potentials observed with potassium gluconate-filled pipettes. Only a 13-mV change in resting potential was observed during similar comparisons in P27-28 neurons. 5. Outward currents activated by depolarization were examined with the use of voltage-clamp techniques in P2-30 neurons. In P2-5 cells, a small, slowly inactivating outward current was evoked with depolarizing commands from holding potentials near -50 mV. By preceding the depolarizing commands with a hyperpolarizing prepulse, an additional early transient outward current was evoked. The sustained and transient outward currents were separated by their kinetic properties and their sensitivity to cobalt (Co2+), TEA, and 4-AP.(ABSTRACT TRUNCATED AT 400 WORDS)
Granule cells acutely dissociated from the dentate gyrus of adult rat brains displayed a single class of high-threshold, voltage-activated (HVA) Ca2+ channels. The kinetics of whole-cell Ca2+ currents recorded with pipette solutions containing an intracellular ATP regenerating system but devoid of exogenous Ca2+ buffers, were fit best by Hodgkin-Huxley kinetics (m2h), and were indistinguishable from those recorded with the nystatin perforated patch method. In the absence of exogenous Ca2+ buffers, inactivation of HVA Ca2+ channels was a predominantly Ca(2+)-dependent process. The contribution of endogenous Ca2+ buffers to the kinetics of inactivation was investigated by comparing currents recorded from control cells to currents recorded from neurons that have lost a specific Ca(2+)-binding protein, Calbindin-D28K (CaBP), after kindling-induced epilepsy. Kindled neurons devoid of CaBP showed faster rates of both activation and inactivation. Adding an exogenous Ca2+ chelator, 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), to the intracellular solution largely eliminated inactivation in both control and kindled neurons. The results are consistent with the hypothesis that endogenous intraneuronal CaBP contributes significantly to submembrane Ca2+ sequestration at a concentration range and time domain that regulate Ca2+ channel inactivation.
A number of calcium buffers were examined for their ability to reduce evoked transmitter release when injected into the presynaptic terminal of the squid giant synapse. Injection of EGTA was virtually ineffective at reducing transmitter release, even at estimated intracellular concentrations up to 80 mM. Conversely, the buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), which has an equilibrium affinity for calcium similar to that of EGTA at pH 7.2, produced a substantial reduction in transmitter release when injected presynaptically. This effect of BAPTA was reversible, presumably because the buffer diffused out of the terminal and into uninjected regions of the presynaptic axon. BAPTA derivatives with estimated intracellular calcium dissociation constants (Kd) ranging from 0.18 to 4.9 microM were effective at reducing transmitter release at similar estimated concentrations. A BAPTA derivative with an estimated intracellular Kd of 31 mM was less effective. BAPTA did not affect presynaptic action potentials or calcium spikes in ways that could explain its ability to reduce transmitter release. The relative effects of presynaptic injections of BAPTA and derivatives are consistent with the calcium-buffering capability of these compounds if the presynaptic calcium transient that triggers release is hundreds of microM or larger. The superior potency of BAPTA compared to EGTA apparently results from the faster calcium-binding kinetics of BAPTA and suggests that the calcium-binding molecule that triggers release binds calcium in considerably less than 200 microsec and is located very close to calcium channels.
The apoplexy of Hippocrates' time remains with us today, un- changed and untreated. We call this syndrome of acute brain damage "stroke"; we know that it most commonly reflects lo- calized tissue hypoxia attributable to reduced blood flow (isch- emia). Focal hypoxia-ischemia also occurs in such contexts as traumatic insults, or cerebral hemorrhages, while global hypox- ia-ischemia occurs in cardiac arrest, near-drowning, and carbon monoxide poisoning. The centuries since Thomas Willis, Jo- hann Wepfer, and Giovanni Morgagni have brought precise definition of cerebral vascular anatomy and the neurological consequences of focal brain lesions, permitting full comprehen- sion of functional deficits; we can prognosticate with sad ac- curacy. But the medical management of stroke patients in 1990 is still the management of symptoms and associated conditions. Despite its status as a major worldwide cause of death and disability, we are no more able than Hippocrates to treat cerebral hypoxia itself. Nevertheless, hope for the development of effective therapy has endured, and in the last few years has been encouraged by the emergence of some promising strategies for reducing the brain's intrinsic susceptibility to hypoxic insults. These tissue- level approaches, sometimes referred to as "parenchymal" ap- proaches to distinguish them from other strategies aimed at influencing blood how, are based on recent information sug- gesting that central neurotransmitter mechanisms, especially those related to the excitatory neurotransmitter glutamate, may play an important role in the pathogenesis of hypoxic neuronal death (Meldrum, 1985; Rothman and Olney, 1986; Choi, 1988b). In this essay I will comment on the possibility of new therapies for cerebral hypoxia directed at glutamate-mediated injury mechanisms, and will briefly mention some other potential ap- proaches. Glutamate and hypoxic neuronal injury The brain is critically dependent on its blood flow for a contin- uous supply of oxygen and glucose. The oscillations of the elec- troencephalogram cease within seconds of cardiac arrest, and only a few minutes of severe ischemia can induce the selective degeneration of certain neuronal populations, including pyram- idal neurons in the CA1 region of the hippocampal formation, striatal medium-sized neurons, neocortical neurons in layers 3,
The neuroprotective effects of dizocilipine maleate (MK-801), a noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) receptor/channel, were tested in the 4-vessel occlusion rat model of forebrain ischemia. Adult Wistar rats, treated intraperitoneally with MK-801 or saline using several different treatment paradigms were subjected to 5 (n = 208) or 15 (n = 62) min of severe, transient forebrain ischemia. In saline-treated animals, 15 min of ischemia (n = 13) produced extensive and consistent loss of pyramidal neurons in the CA1 zone of hippocampus. The degree and distribution of cell loss were not reduced by single dose preischemic administration of MK-801 at 1 (n = 7), 2.5 (n = 4), or 5 mg/kg (n = 8). In other animals subjected to 15 min of forebrain ischemia, multiple doses of MK-801 (5, 2.5, and 2.5 mg/kg) given immediately and at approximately 8 and 20 hr after cerebral reperfusion (n = 5) did not alter CA1 injury compared to saline-treated controls (n = 5). Five minutes of forebrain ischemia in saline-treated animals, (n = 82) resulted in significantly fewer (p less than 0.001) dead CA1 pyramidal cells and a greater variance compared to animals subjected to 15 min of ischemia. Power analysis of the preliminary saline-treated animals subjected to 5 min of ischemia (n = 22) indicated that 60 animals per group were necessary to detect a 15% difference between MK-801 and vehicle-treated groups. Multidose treatment with MK-801 (1 mg/kg) given 1 hr prior to 5 min of ischemia (n = 60) and again at approximately 8 and 16 hr after recirculation failed to attenuate hippocampal injury.(ABSTRACT TRUNCATED AT 250 WORDS)
The relationship between neuronal calcium binding protein content (calbindin D28K: CaBP and parvalbumin: PV) and vulnerability to ischemia was studied in different regions of the rat brain using the four vessel occlusion model of complete forebrain ischemia. The areas studied, i.e. the hippocampal formation, neocortex, neostriatum and reticular thalamic nucleus (RTN), show a characteristic pattern of CaBP and PV distribution, and are involved in ischemic damage to different degrees. In the hippocampal formation CaBP is present in dentate granule cells and in a subpopulation of the CA1 pyramidal cells, the latter being the most and the former the least vulnerable to ischemia. Non-pyramidal cells containing CaBP in these regions survive ischemia, whereas PV-containing non-pyramidal cells in the CA1 region are occasionally lost. Hilar somatostatin-containing cells and CA3 pyramidal cells contain neither PV nor CaBP. Nevertheless, the latter are resistant to ischemia and the former is the first population of cells that undergoes degeneration. Supragranular pyramidal neurons containing CaBP are the most vulnerable cell group in the sensory neocortex. In the RTN the degenerating neurons contain both PV and CaBP. In the neostriatum, ischemic damage involves both CaBP-positive and negative medium spiny neurons, although the degeneration always starts in the dorsolateral neostriatum containing relatively few CaBP-positive cells. The giant cholinergic interneurons of the striatum contain neither CaBP nor PV, and they are the most resistant cell type in this area. These examples suggest the lack of a consistent and systematic relationship between neuronal CaBP or PV content and ischemic vulnerability. It appears that some populations of cells containing CaBP or PV are more predisposed to ischemic cell death than neurons lacking these proteins. These neurons may express high levels of calcium binding proteins because their normal activity may involve a high rate of calcium uptake and/or intraneuronal release.
Confocal laser-scanned microscopy and long-wavelength calcium (Ca2+) indicators were combined to monitor both sustained and
rapidly dissipating Ca2+ gradients in voltage-clamped sympathetic neurons isolated from the bullfrog. After a brief activation
of voltage-dependent Ca2+ channels, Ca2+ spreads inwardly, and reaches the center of these spherical cells in about 300 milliseconds.
Although the Ca2+ redistribution in the bulk of the cytosol could be accounted for with a radial diffusion model, local nonlinearities,
suggesting either nonuniform Ca2+ entry or spatial buffering, could be seen. After electrical stimulation, Ca2+ signals in
the nucleus were consistently larger and decayed more slowly than those in the cytosol. A similar behavior was observed when
release of intracellular Ca2+ was induced by caffeine, suggesting that in both cases large responses originate from Ca2+ release
sites near or within the nucleus. These results are consistent with an amplification mechanism involving Ca2(+)-induced Ca2+
release, which could be relevant to activity-dependent, Ca2(+)-regulated nuclear events.
High concentrations of potent N-methyl-D-aspartate (NMDA) agonists can trigger degeneration of cultured mouse cortical neurons
after an exposure of only a few minutes; in contrast, selective non-NMDA agonists or low levels of NMDA agonists require exposures
of several hours to induce comparable damage. The dihydropyridine calcium channel antagonist nifedipine was used to test whether
this slow neurotoxicity is mediated by a calcium influx through voltage-gated channels. Nifedipine had little effect on the
widespread neuronal degeneration induced by brief exposure to high concentrations of NMDA but substantially attenuated the
neurotoxicity produced by 24-hour exposure to submaximal concentrations of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate,
kainate, or quinolinate. Calcium ion influx through dihydropyridine-sensitive, voltage-dependent calcium channels may be an
important step in the neuronal injury induced by the prolonged activation of NMDA or non-NMDA glutamate receptors.
Intracellular free-calcium concentration ([Ca2+]i) was measured in lamprey spinal axons using the fluorescent calcium indicator fura 2. We used both a photomultiplier tube and a video-image processing system to measure the temporal and spatial distributions of [Ca2+]i in the proximal segments of transected axons. Within 3 min following transection, a spatially graded increase in the [Ca2+]i was apparent in the last few millimeters of the axons. Superimposed on the initial gradient was a moving front of calcium that progressed up the axon, reaching 1.6 mm from the cut end in 3 hr. The [Ca2+]i behind the moving front exceeded 10 microM. This movement of Ca2+ was greatly reduced by an externally applied electrical field with the cathode distal to the lesion and was increased by an applied field of the opposite polarity. When axons were transected in Ca2(+)-free medium, no increases in [Ca2+]i occurred. One d after transection, [Ca2+]i was at or below the precut levels, except in the distal 250 microns, where it remained slightly elevated. Therefore, axons can survive the high levels of [Ca2+]i that occur after transection and can reestablish normal [Ca2+]i levels within 24 hr. Measurements of both the diffusion coefficient and the fluorescence polarization of fura 2 indicate that the dye is not significantly bound to axoplasmic components.
We have developed a calcium diffusion model for a spherical neuron which incorporates calcium influx and extrusion through the plasma membrane as well as three calcium buffer systems with different capacities, mobilities, and kinetics. The model allows us to calculate the concentration of any of the species involved at all locations in the cell and can be used to account for experimental data obtained with high-speed Ca imaging techniques. The influence of several factors on the Ca2+ transients is studied. The relationship between peak [Ca2+]i and calcium load is shown to be nonlinear and to depend on buffer characteristics. The time course of the Ca2+ signals is also shown to be dependent on buffer properties. In particular, buffer mobility strongly determines the size and time course of Ca2+ signals in the cell interior. The model predicts that the presence of exogenous buffer, such as fura-2, modifies the Ca2+ transients to a variable extent depending on its proportion relative to the natural, intrinsic buffers. The conclusions about natural calcium buffer properties that can be derived from Ca imaging experiments are discussed.
Several laboratories have reported a significant reduction of ischemia-induced injury to hippocampal neurons in rodents treated with competitive and noncompetitive N-methyl-D-aspartate (NMDA) receptor-channel antagonists. This study examined the effects of the noncompetitive antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) in Mongolian gerbils subjected to 5 min of bilateral carotid artery occlusion. In adult female gerbils, single doses of MK-801 injected 1 hr prior to ischemia significantly (p less than 0.01) reduced damage to CA1 hippocampal neurons. However, the drug rendered the postischemic animals comatose and hypothermic for several hours compared with the saline-treated animals. In subsequent experiments, animals pretreated with MK-801 and maintained normothermic during and after forebrain ischemia demonstrated no amelioration of hippocampal damage. Gerbils not treated with MK-801, but kept hypothermic in the postischemic period to approximately the same degree (34.5 degrees C) and duration (8 hr) as was induced by MK-801 therapy showed significant (p less than 0.01) protection of CA1 neurons against ischemia. The neuroprotective activity of MK-801 against transient global ischemia appears to be largely a consequence of postischemic hypothermia rather than a direct action on NMDA receptor-channels.
The neurotoxicity of 3 non-NMDA glutamate receptor agonists--kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA), and quisqualate--was investigated quantitatively in dissociated murine cortical cultures. Five minute exposure to 500 microM kainate, but not AMPA, produced widespread acute neuronal swelling. Kainate-induced swelling was resistant to 2-amino-5-phosphonovalerate (APV) or replacement of extracellular sodium with choline but attenuated by either kynurenate or low concentrations of quisqualate. Unlike NMDA agonists, kainate or AMPA did not produce much late neuronal loss after a 5 min exposure. In contrast, 5 min exposure to 500 microM quisqualate produced both acute neuronal swelling and widespread late neuronal degeneration. This acute swelling was blocked by APV or by replacement of extracellular sodium by choline, consistent with mediation by NMDA receptors; we speculate that high concentrations of quisqualate may directly activate NMDA receptors or induce the release of endogenous glutamate. Quisqualate-induced late neuronal degeneration may be due to another unexpected process: cellular quisqualate uptake and delayed release, converting brief addition into prolonged exposure. Hours after thorough washout of exogenously added quisqualate, micromolar concentrations could be detected in the bathing medium by high performance liquid chromatography. With lengthy exposure (20-24 hr), all 3 non-NMDA agonists were potent neurotoxins, able to destroy neurons with EC50's of about 20 microM for kainate, 4 microM for AMPA, and 1 microM for quisqualate. Kynurenate and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but not APV or L-glutamate diethyl ester, were effective in attenuating the neuronal degeneration induced by these agonists. CNQX was about 3 times more selective than kynurenate against kainate-induced neuronal injury, but CNQX was still nearly equipotent with APV against NMDA-induced injury. Gamma-D-glutamylaminomethyl sulfonate exhibited partial antagonist specificity for AMPA-induced toxicity.
: Using primary cultures of cerebral cortical neurons, it has been demonstrated that the antihyperthermia drug dantrolene completely protects against glutamate-induced neurotoxicity. Furthermore, in the presence of extracellular calcium, dantrolene reduced the glutamate-induced increase in the intracellular calcium concentration by 70%. In the absence of extracellular calcium, this glutamate response was completely blocked by dantrolene. Dantrolene did not affect the kinetics of [3H]glutamate binding to membranes prepared from similar cultures. These results indicate that release of calcium from intracellular stores is essential for the propagation of glutamate-induced neuronal damage. Because it is likely that glutamate is involved in neuronal degeneration associated with ischemia and hypoxia, the present findings might suggest that dantrolene and possibly other drugs affecting intracellular calcium pools might be of therapeutic interest.
The cytoprotective effect of NBQX, a selective AMPA receptor antagonist, was tested following 10 min of severe forebrain ischemia using the 4-vessel occlusion model. Immediately, and at 15 and 30 min following reperfusion, adult Wistar rats received intraperitoneal injections of either saline (n = 5), 1 mg lithium chloride (n = 17) or 30 mg/kg of the lithium salt of NBQX (n = 18). In saline-treated animals 82 ± 12% of CA1 hippocampal neurons were lost. Of those treated with lithium 70 ± 23% were injured, while those given NBQX sustained only 40 ± 34% CA1 necrosis (P < 0.01). Twelve of 18 NBQX-treated animals had less than 30% CA1 injury as compared with 1 of 17 lithium-treated animals. The AMPA receptor may play a more important role than the NMDA receptor in selective ischemic necrosis of hippocampal neurons.
The quinoxalinedione, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), has been introduced as a relatively selective antagonist of non-N-methyl-d-aspartate (non-NMDA) glutamate receptors. We studied the ability of CNQX to block excitatory amino acid-induced neurotoxicity in murine cortical cell cultures. 100 μM CNQX blocked the acute neuronal swelling induced by 500 μM kainate, but it also attenuated the swelling and degeneration induced by 500 μM NMDA. Addition of 1 mM glycine to the CNQX eliminated antagonism of NMDA toxicity while preserving antagonism of the neuronal degeneration induced by kainate or AMPA. This selective non-NMDA antagonist combination of CNQX plus glycine substantially attenuated the acute neuronal swelling induced by brief exposure to 500 μM glutamate, but had little effect on subsequent late degeneration, supporting the conclusion that rapidly triggered glutamate-induced cortical neuronal death is predominantly mediated by NMDA receptors.
The function of calcium entry or release channels is often modulated by the cytosolic free calcium concentration. When such channels are studied in isolation, calcium buffer solutions are usually used to control the free calcium at the cytosolic face of the channel. Such solutions are generally formulated on the basis of equilibrium considerations. We calculate the gradlent of [Ca2+] in the vicinity of a channel pore, in the presence of such buffers. We find that the effective degree of buffering near the pore is markedly affected by kinetic considerations. Commonly used EGTA solutions are completely ineffective in buffering [Ca2+] within macromolecular distances of the pore. In order to achieve useful buffering, the fastest buffers (e.g. BAPTA derivatives) must be used, in concentrations very much higher than those conventionally employed. Because of the diffusion limit on the maximum rate of binding of calcium to the buffer ligand, it is physically impossible to achieve good control of [Ca2+] at cytosolic levels at distances of less than a few nm from a pore conducting pico-ampere calcium current.
The function of calcium entry or release channels is often modulated by the cytosolic free calcium concentration. When such channels are studied in isolation, calcium buffer solutions are usually used to control the free calcium at the cytosolic face of the channel. Such solutions are generally formulated on the basis of equilibrium considerations. We calculate the gradient of [Ca2+] in the vicinity of a channel pore, in the presence of such buffers. We find that the effective degree of buffering near the pore is markedly affected by kinetic considerations. Commonly used EGTA solutions are completely ineffective in buffering [Ca2+] within macromolecular distances of the pore. In order to achieve useful buffering, the fastest buffers (e.g. BAPTA derivatives) must be used, in concentrations very much higher than those conventionally employed. Because of the diffusion limit on the maximum rate of binding of calcium to the buffer ligand, it is physically impossible to achieve good control of [Ca2+] at cytosolic levels at distances of less than a few nm from a pore conducting pico-ampere calcium current.
Large neurons from layer V in a slice preparation of cat sensorimotor cortex were impaled with microelectrodes containing KCl plus different concentrations of the Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA) or two of its derivatives. Impalement with electrodes containing high BAPTA (200 mM) quickly abolished Ca(2+)-dependent afterhyperpolarizations. Spike parameters were normal, but the usual time- and voltage-dependent rectification of subthreshold membrane potential was absent. Normally, this rectification results from activation of two voltage-gated currents, the persistent sodium current (INaP) and the hyperpolarizing inward rectifier current (Ih). Both of these currents were absent during voltage clamp with high BAPTA microelectrodes. Impalement with electrodes containing low BAPTA (2 mM) or derivatives caused a different effect. Injection of a 1-s current pulse evoked phasic firing instead of the tonic firing seen normally. Both the amplitude and the duration of the Ca(2+)-dependent afterhypolarization that followed repetitive firing were much greater than normal. The effectiveness of BAPTA derivatives in altering afterhyperpolarizations and firing properties were similar to their effectiveness in chelating Ca2+. It is assumed that the BAPTA effects result from reduction of intracellular Ca2+ concentration. Results with high BAPTA suggest that (i) both INaP and Ih require a minimal intracellular calcium concentration for normal expression, and that (ii) these voltage-gated currents may be modulated by changes in intracellular calcium concentration. Results with low BAPTA suggest that a small reduction of intracellular calcium concentration preferentially enhances a slow, Ca(2+)-dependent K+ current which then dominates the firing properties of the cell. The transformed firing properties resemble those of hippocampal pyramidal neurons.(ABSTRACT TRUNCATED AT 250 WORDS)
Previous work demonstrating the presence and differential distribution of Ca(2+)-binding proteins in the CNS has led to the proposal that cytosolic proteins, such as calbindin-D28k (CB), may play a pivotal role in neurons. We have used a retrovirus containing the full-length cDNA for CB to transfect the pituitary tumor cell line GH3, to generate CB-expressing GH3 cells and to investigate whether ionic channel activities as well as the concentration of intracellular free Ca2+ ([Ca2+]i) homeostasis could be altered by the presence of this Ca(2+)-binding protein. We show that CB-transfected GH3 cells exhibited lower Ca2+ entry through voltage-dependent Ca2+ channels and were better able to reduce [Ca2+]i transients evoked by voltage depolarizations than the wild-type parent cell line. These observations provide a mechanism by which CB may protect tissues against Ca(2+)-mediated excitotoxicity.
1. Digital imaging and photometry were used in conjunction with the fluorescent Ca2+ indicator, Fura-2, to examine intracellular Ca2+ signals produced by depolarization of single adrenal chromaffin cells. 2. Depolarization with a patch pipette produced radial gradients of Ca2+ within the cell, with Ca2+ concentration highest in the vicinity of the plasma membrane. These gradients dissipated within a few hundred milliseconds when the voltage-gated Ca2+ channels were closed. 3. Dialysis of Fura-2 into the chromaffin cell caused concentration-dependent changes in the depolarization-induced Ca2+ signal, decreasing its magnitude and slowing its recovery time course. These changes were used to estimate the properties of the endogenous cytoplasmic Ca2+ buffer with which Fura-2 competes for Ca2+. 4. The spatially averaged Fura-2 signal was well described by a model assuming fast competition between Fura-2 and an endogenous buffer on a millisecond time scale. Retrieval of calcium by pumps and slow buffers occurs on a seconds-long time scale. No temporal changes indicative of buffers with intermediate kinetics could be detected. 5. Two independent estimates of the capacity of the fast endogenous Ca2+ buffer suggest that 98-99% of the Ca2+ entering the cell normally is taken up by this buffer. This buffer appears to be immobile, because it does not wash out of the cell during dialysis. It has a low affinity for Ca2+ ions, because it does not saturate with 1 microM-Ca2+ inside the cell. 6. The low capacity, affinity and mobility of the endogenous Ca2+ buffer makes it possible for relatively small amounts of exogenous Ca2+ buffers, such as Fura-2, to exert a significant influence on the characteristics of the Ca2+ concentration signal as measured by fluorescence ratios. On the other hand, even at moderate Fura-2 concentrations (0.4 mM) Fura-2 will dominate over the endogenous buffers. Under these conditions radiometric Ca2+ concentration signals are largely attenuated, but absolute fluorescence changes (at 390 nm) accurately reflect calcium fluxes.
Among the many calcium-binding proteins in the nervous system, parvalbumin, calbindin-D28K and calretinin are particularly striking in their abundance and in the specificity of their distribution. They can be found in different subsets of neurons in many brain regions. Although it is not yet known whether they play a 'triggering' role like calmodulin, or merely act as buffers to modulate cytosolic calcium transients, they are valuable markers of neuronal subpopulations for anatomical and developmental studies.
We describe a new rat model of temporary focal ischemia that produces neocortical ischemia without the need for prolonged anesthesia.
Temporary focal cerebral ischemia was initiated during halothane anesthesia, maintained for varying periods without anesthesia, and reversed by clip removal requiring brief anesthesia. Tandem carotid and middle cerebral artery occlusion for 1-4 hours and permanent occlusion were used to determine the duration and extent of ischemia necessary to produce predictable volumes of neocortical infarction in Wistar and spontaneously hypertensive rats.
In Wistar rats, occlusion of the right middle cerebral and both common carotid arteries resulted in cerebral blood flow reductions to approximately 8% of baseline. One hour of transient ischemia with 23 hours of reperfusion did not result in infarction. Three hours of ischemia followed by 21 hours of reperfusion resulted in infarction comparable to that caused by 24 hours of permanent ischemia. In spontaneously hypertensive rats, unilateral right middle cerebral and common carotid artery occlusion reduced cerebral blood flow to approximately 11% of baseline. Minimal damage was seen with 1 hour of reversible ischemia, but intervals of 2 and subsequently 3 hours followed by 22-21 hours of reperfusion produced progressively larger infarcts. Damage indistinguishable from that seen with 24 hours of permanent ischemia was seen with 3 or 4 hours of transient ischemia followed by 21 or 20 hours of reperfusion.
For unanesthetized normothermic rats, cerebral blood flow reductions to 10-20% of baseline resulted in maximal infarction once ischemic durations exceeded 2-3 hours. To be effective, experimental therapies aimed at lessening infarct size or restoring blood flow must be initiated within this critical time interval.
The relation between time-dependent changes in cerebral blood flow and the appearance of infarction after focal cerebral ischemia is still a matter for debate. The aim of this study was to measure perfusion after simultaneous occlusions of the left middle cerebral artery and ipsilateral common carotid artery in rats and correlate it with the timing and distribution of histological changes.
We studied histological and cerebral blood flow changes 5 minutes and 4, 24, and 48 hours after the onset of focal ischemia. Blood flow was determined autoradiographically using [14C]iodoantipyrine. A coronal template subdivided into regions of interest was applied to the autoradiographs and the histological data.
In some regions of the nonoccluded hemisphere, cerebral blood flow 5 minutes after occlusion fell below 50% of normal. Many ischemic structures showed stable blood flow for 48 hours after occlusion, confirming that in this model reperfusion is minimal. Infarction occurred eventually in all areas in which blood flow at 5 minutes fell below 10% of that in control rats, but infarction appeared earlier in regions in which blood flow at 5 minutes was below 5% of that in control rats. When blood flow at 5 minutes rose above 12% of that in control rats, the occurrence of infarction became unpredictable.
Despite the general dependence of infarction on perfusion levels, blood flow was not a reliable indicator of those regions committed to infarction.
When applied to rat hippocampal slices, the permeable calcium chelator, BAPTA-AM, caused a reduction of both post-spike train slow afterhyperpolarizations (AHPs) and spike-frequency adaptation in dentate granule cells. This indicated that BAPTA-AM can, like microinjected EGTA, block calcium-activated potassium channels. At perforant pathway synapses, BAPTA-AM caused a reduction of inhibitory postsynaptic potentials (IPSPs) and an initial increase and later decrease of excitatory postsynaptic potentials (EPSPs). The initial increase in EPSPs may be caused by presynaptic spike-broadening owing to inhibition of calcium-activated potassium channels which normally regulate the duration of the presynaptic action potential. These channels may be affected at lower doses of chelator than synaptic transmitter release. BAPTA salt injected into individual dentate granule cells caused, as expected, decreased AHPs and spike-frequency adaptation. Also, paradoxically, both excitatory and inhibitory synaptic potentials were increased although input resistance was not.
Excitatory amino acids and their receptors play an important role in membrane phospholipid metabolism. Persistent stimulation of excitatory amino acid receptors by glutamate may be involved in neurodegenerative diseases and brain and spinal cord trauma. The molecular mechanism of neurodegeneration induced by excitatory amino acids is, however, not known. Excitotoxin induced calcium entry causes the stimulation of phospholipases and lipases. These enzymes act on neural membrane phospholipids and their stimulation results in accumulation of free fatty acids, diacylglycerols, eicosanoids and lipid peroxides in neurodegenerative diseases and brain and spinal cord trauma. Other enzymes such as protein kinase C and calcium-dependent proteases may also contribute to the neuronal injury. Excitotoxin-induced alteration in membrane phospholipid metabolism in neurodegenerative diseases and neural trauma can be studied in animal and cell culture models. The models can be used to study the molecular mechanisms of the neurodegenerative processes and to screen the efficacy of therapeutic drugs for neurodegenerative disease and brain and spinal cord trauma.
Neuronal systems for calcium homeostasis are crucial for neuronal development and function and may also contribute to selective neuronal vulnerability in adverse conditions such as exposure to excitatory amino acids or anoxia, and in neurodegenerative diseases. Previous work demonstrated the presence and differential distribution of calcium-binding proteins in the CNS. We now report that a subpopulation of neurons in dissociated cell cultures of embryonic rat hippocampus expresses calbindin-D28k (Mr 28,000 calcium-binding protein) immunoreactivity and that these neurons are relatively resistant to neurotoxicity induced by either glutamate or calcium ionophore. Direct comparisons of dynamic aspects of intracellular calcium levels and calbindin-D28k immunoreactivity in the same neurons revealed that calbindin-D28k-positive neurons were better able to reduce free intracellular calcium levels than calbindin-D28k-negative neurons. These findings indicate that the differential expression of calbindin-D28k in hippocampal neurons occurs early in development and may be one determinant of selective neuronal vulnerability to excitotoxic insults.
We assessed the pathways by which excitatory and inhibitory neurotransmitters elicit postsynaptic changes in [Ca2+]i in brain slices of developing rat and cat neocortex, using fura 2. Glutamate, NMDA, and quisqualate transiently elevated [Ca2%]i in all neurons. While the quisqualate response relied exclusively on voltage-gated Ca2+ channels, almost all of the NMDA-induced Ca2+ influx was via the NMDA ionophore itself, rather than through voltage-gated Ca2+ channels. Glutamate itself altered [Ca2+]i almost exclusively via the NMDA receptor. Furthermore, synaptically induced Ca2+ entry relied almost completely on NMDA receptor activation, even with low-frequency stimulation. The inhibitory neurotransmitter GABA also increased [Ca2+]i, probably via voltage-sensitive Ca2+ channels, whereas the neuromodulator acetylcholine caused Ca2+ release from intracellular stores via a muscarinic receptor. Low concentrations of these agonists produced nonperiodic [Ca2+]i oscillations, which were temporally correlated in neighbouring cells. Optical recording with Ca2(+)-sensitive indicators may thus permit the visualization of functional networks in developing cortical circuits.
The neurotoxicity of glutamate was investigated quantitatively in mixed neuronal and glial spinal cord cell cultures from fetal mice at 12-13 days of gestation. Five-minute exposure to 10-1000 microM glutamate produced widespread acute neuronal swelling, followed by neuronal degeneration over the next 24 h (EC50 for death about 100-200 microM); glia were not injured. Glutamate was neurotoxic in cultures as young as four days in vitro, although greater death was produced in older cultures. By 14-20 days in vitro, 80-90% of the neuronal population was destroyed by a 5-min exposure to 500 microM glutamate. Acute neuronal swelling following glutamate exposure was prevented by replacement of extracellular sodium with equimolar choline, with minimal reduction in late cell death. Removal of extracellular calcium enhanced acute neuronal swelling but attenuated late neuronal death. Both acute neuronal swelling and late degeneration were effectively blocked by the noncompetitive N-methyl-D-aspartate receptor antagonist dextrorphan and by the novel competitive antagonist CGP 37849. Ten micromolar 7-chlorokynurenate also inhibited glutamate neurotoxicity; protection was reversed by the addition of 1 mM glycine to the bathing medium. These observations suggest that glutamate is a potent and rapidly acting neurotoxin on cultured spinal cord neurons, and support involvement of excitotoxicity in acute spinal cord injury. Similar to telencephalic neurons, spinal neurons exposed briefly to glutamate degenerate in a manner dependent on extracellular Ca2+ and the activation of N-methyl-D-aspartate receptors.
Focal cerebral ischemia was produced by occluding the left middle cerebral artery in 769 rats. Permeability of the blood-brain barrier to small or large molecules was evaluated qualitatively using Evans blue or sodium fluorescein and quantitatively using the transfer indexes of iodine-125-labeled bovine serum albumin or [14C]sucrose. Water content was determined using wet and dry weights and sodium and potassium contents using flame photometry. Cortical tissue in the middle cerebral artery territory was sampled less than or equal to 14 days after occlusion. A significant increase in the albumin transfer index was first found 12 hours after occlusion, and the index remained approximately the same until water content peaked 3 days after occlusion. In contrast, the sucrose transfer index increased gradually, significantly correlated with increases in the water and sodium contents. Tissue staining by sodium fluorescein was more extensive than that by Evans blue. As edema fluid decreased gradually 4-10 days after occlusion, the albumin and sucrose transfer indexes increased markedly. These findings indicate that disruption of the blood-brain barrier to small molecules is accompanied by accumulation of edema fluid during the later stages of ischemia. Opening of the barrier to serum protein is probably related to the resolution of edema.
Accurate and reproducible determination of the size and location of cerebral infarcts is critical for the evaluation of experimental focal cerebral ischemia. The purpose of this study was to compare intracardiac perfusion of 2,3,5-triphenyltetrazolium chloride with immersion of brain tissue in 2,3,5-triphenyltetrazolium chloride to delineate brain infarcts in rats.
After 6, 24, or 48 hours of ischemia induced by permanent middle cerebral artery occlusion, some rats were perfused with 2,3,5-triphenyltetrazolium chloride; other rats were given an overdose of barbiturates, after which brain sections were immersed in 2,3,5-triphenyltetrazolium chloride. Coronal sections were taken 4, 6, and 8 mm from the frontal pole, and infarct areas in perfused and immersed sections were compared; subsequently, the same sections were stained with hematoxylin and eosin.
In rats subjected to 24 or 48 hours of occlusion, areas of infarction were clearly defined with both 2,3,5-triphenyltetrazolium chloride staining techniques, and the infarct sizes correlated well with the results of hematoxylin and eosin staining (r = 0.85-0.94).
These results demonstrate that intracardiac perfusion of 2,3,5-triphenyltetrazolium chloride is an accurate, inexpensive, and efficient staining method to detect infarcted tissue 24 and 48 hours after the onset of ischemia in rats.
We report the regional variation in [3H]nimodipine binding in vivo during focal cerebral ischemia. After intravenous injection, 30 min of circulation of [3H]nimodipine was sufficient to establish a secular equilibrium of distribution in the brain. Rats sustained left middle cerebral and common carotid artery occlusions for 5 min, and 4, 24, and 48 h (n greater than or equal to 6 epr group). They were decapitated 30 min after injection of 250 microCi of [3H]nimodipine and their brains were submitted to autoradiography. The concentrations of [3H]nimodipine in plasma and brain structures, corrected for metabolism of nimodipine, were used to calculate the regional volumes of distribution (V) in the ischemic left (L) and control right (R) hemispheres. Log (VL/VR) was then defined as the group mean of the logarithms of the left-to-right ratio of V of [3H]nimodipine. In the lateral caudate, binding of [3H]nimodipine on the ischemic side was highest within 5 min of occlusion. Log (VL/VR) in this region for the combined sham-operated and normal control rats and those after 5 min and 4 and 24 h of ischemia were -0.014 +/- 0.025, 0.137 +/- 0.056*, -0.201 +/- 0.367, and -0.049 +/- 0.370 (mean +/- SD, *represents p less than 0.01 compared with controls). By contrast, in the superior frontal cortex, values for log (VL/VR) in the same sequence were -0.016 +/- 0.025, 0.028 +/- 0.056, 0.284 +/- 0.228*, and 0.224 +/- 0.069*, thus showing a significant rise in [3H]nimodipine binding only at 4 h.(ABSTRACT TRUNCATED AT 250 WORDS)
We exposed murine cortical neuronal cell cultures for 24 hours to defined concentrations of N-methyl-D-aspartate (NMDA), kainate, or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and assessed the resultant neuronal degeneration quantitatively by the efflux of lactate dehydrogenase to the bathing medium. The small subpopulations of neurons that stained immunohistochemically for either somatostatin- or parvalbumin-like reactivity were atypically affected by these excitotoxins. Limited exposure to kainate or AMPA did little damage to the general neuronal population, but destroyed nearly all somatostatin- or parvalbumin-reactive cells. Conversely, these immunoreactive cells were more resistant to NMDA-induced injury than the general population. In view of reports suggesting that somatostatin- and parvalbumin-reactive cortical neurons may be preferentially damaged in Alzheimer's disease, these observations support a hypothesis that the overactivation of non-NMDA receptors could be involved in Alzheimer's disease pathogenesis.
Quin2-acetoxymethylester (quin2/AM) (50 microM), administered directly to the motoneuronal pool of the frog spinal cord, could be loaded into the motoneuron as well as the other cells in the lumbar region. Depolarizing responses of the ventral root to L-glutamate in the quin2-loaded side persisted even after prolonged exposure to A23187 (2.0 microM), while the responses in the unloaded side were markedly reduced. Histologically confirmed neuronal cell loss from the motoneuronal pool induced by A23187 (2.0 microM) or by a high concentration of L-glutamate (10 mM) was prevented by pretreatment with quin2/AM. A23187- and L-glutamate-induced histological and functional damage in neuronal cells and the protective effects of quin2 on them provide further evidence for cell death due to Ca2+ overloading.
Cytotoxic overactivation of neuronal N-methyl-D-aspartate receptors is postulated to contribute to the pathogenesis of neuronal loss in hypoxia-ischemia. However, several events associated with hypoxia-ischemia may limit N-methyl-D-aspartate receptor-mediated injury. Perhaps the most important of these events is the development of extracellular acidosis. These limiting events may help explain why N-methyl-D-aspartate receptors contribute to injury more in focal ischemia or hypoxia in vitro than in global ischemia.
2,3-Dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) is an analog of the quinoxalinedione antagonists to the non-N-methyl-D-aspartate (non-NMDA) glutamate receptor. NBQX is a potent and selective inhibitor of binding to the quisqualate subtype of the glutamate receptor, with no activity at the NMDA and glycine sites. NBQX protects against global ischemia, even when administered 2 hours after an ischemic challenge.
An excitotoxic action of glutamate and aspartate contributes to the pathological outcome after transient global cerebral ischaemia, focal ischaemia, neonatal hypoxia/ischaemia, and secondary ischaemia following brain trauma. This provides a therapeutic approach utilising drugs acting on (i) glutamate release, (ii) postsynaptic glutamate receptors, and (iii) the secondary events following receptor activation (including the arachidonic acid cascade). Both NMDA and non-NMDA receptors are involved in the excitotoxic effects of glutamate and aspartate. The availability of competitive and noncompetitive antagonists acting at the NMDA receptor has permitted the demonstration of cerebroprotective effects of these compounds in animal models of global, focal, neonatal, and secondary cerebral ischaemia. Protection is seen with antagonist administration prior to and after the onset of ischaemia. The postischaemic therapeutic time window is not fully defined for the different models but is in the range of 0-20 min for incomplete global ischaemia and 1-3 h for focal ischaemia. The clinical usefulness of this approach remains to be established.
Pancreatic acini were loaded with the Ca-selective fluorescent indicator quin-2 by incubation with its acetyoxymethyl ester. Loading acini with 844 +/- 133 microM quin-2 altered neither their ultrastructure nor their viability. The rate of amylase release from quin-2-loaded acini in response to the secretagogue carbachol, however, was significantly smaller than that of control acini. Studies in which acini were loaded with both quin-2 and a similar Ca-chelating compound, BAPTA, indicated that this reduced amylase release was related to the Ca buffering properties of quin-2. The concentration of free intracellular Ca calculated from the fluorescence of quin-2 was 90 +/- 18 nM. Stimulation by carbachol of acini suspended in media containing 1.25 mM Ca caused a rapid, transient enhancement of this value. After stimulation amylase release, the onset of the rise in free cytosolic Ca levels was observed in 1.1 +/- 0.1 s following the addition of agonist, and peak Ca levels (545 +/- 112 nM) were obtained within 5.3 +/- 0.3 s. For concentrations of carbachol less than or equal to 10(-6) M, a stoichiometric relation was found between stimulated amylase release and the peak concentration of free cytosolic Ca achieved. At higher concentrations of carbachol, however, the peak free cytosolic Ca remained constant while amylase release declined. The latency of the rise in intracellular Ca following stimulation of acini suspended in Ca-free media was not different from that observed for acini suspended in normal media, but the rise time was significantly prolonged. In the presence of extracellular Ca, the intracellular level of Ca remained elevated 2.8-fold above basal levels for at least 15 min following stimulation with 10(-6) M carbachol, whereas it had returned to near resting levels by 15 min when either 3 X 10(-7) or 3 X 10(-5) M carbachol was the stimulus. The Ca ionophore ionomycin (10-6 M) induced changes in the level of free cytosolic Ca similar to those caused by 10(-6) M carbachol. Ionomycin, however, stimulated only approximately one-third as much amylase release. These data suggest that factors in addition to changes in free cytosolic Ca may be important in regulating enzyme secretion by pancreatic acinar cells.
K channels of bovine adrenal chromaffin cells were studied using patch-clamp techniques. Whole-cell K currents measured near +10 mV were much larger in 1 mM-external Ca than in Ca-free saline. Noise analysis suggested that this Ca-dependent current was carried by a large unitary conductance channel, called BK channel, which was previously described in inside-out patches (Marty, 1981). The Ca-dependent K current near +10 mV declined with time due to 'run-down' of Ca channels. At the same time, a fraction of the outward current observed above +50 mV was also eliminated. This outward current component probably represents K efflux through Ca channels. Whole-cell Ca-dependent K currents were studied using various Ca buffers. EGTA buffers were surprisingly inefficient: in order to block the current entirely, it was necessary to use an isotonic EGTA solution and to increase internal pH. 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was at least five times more efficient than EGTA. In isolated patches three types of single-channel K currents were observed. Under normal ionic conditions (140 mM-K inside, 140 mM-Na outside), the unitary conductances measured between -20 and +40 mV were 96 pS, 18 pS and 8 pS. The 96 pS channels are the Ca-dependent BK channels. 18 pS and 8 pS channels were both activated and then inactivated by membrane depolarization. Both displayed complex kinetics; single-channel currents were grouped in bursts. Activation and inactivation kinetics were faster for the 18 pS channel (therefore termed FK channel, for fast K channel) than for the 8 pS channel (SK channel, for slow or small amplitude channel). The voltage dependence of opening probability was steeper for the FK channel as compared to the SK channel.
We have evaluated the use of 2,3,5-triphenyltetrazolium chloride (TTC) as an histopathologic stain for identification of infarcted rat brain tissue. The middle cerebral artery (MCA) of 35 normal adult rats was occluded surgically. At various times after surgical occlusion, rats were sacrificed and brain slices were obtained and stained with TTC or hematoxolin and eosin (H & E); the size of the area of infarcted tissue stained by each method was quantified. In rats sacrificed 24 hr after occlusion of the MCA, the size of the area of infarction was 21 +/- 2% of the coronal section for TTC, and 21 +/- 2% for H & E (mean +/- S.D., N = 13). The size of areas of infarction determined by either staining method was not significantly different in area by the paired test, and a significant correlation between sizes determined by each method was found by linear regression analysis (r = 0.91, slope = 0.89, and the y intercept = 4.4%). Staining with TTC is a rapid, convenient, inexpensive, and reliable method for the detection and quantification of cerebral infarction in rats 24 hr after the onset of ischemia.