Oligodendrocyte degeneration and recovery after focal cerebral ischemia.

The vulnerability of oligodendrocytes to ischemic injury may contribute to functional loss in diseases of central white matter. Immunocytochemical methods to identify oligodendrocyte injury in experimental models rely on epitope availability, and fail to discriminate structural changes in oligodendrocyte morphology. We previously described the use of a lentiviral vector (LV) carrying enhanced green fluorescent protein (eGFP) under the myelin basic protein (MBP) promoter for selective visualization of oligodendrocyte cell bodies and processes. In this study, we used LV-MBP-eGFP to label oligodendrocytes in rat cerebral white matter prior to transient focal cerebral ischemia, and examined oligodendrocyte injury 24 h, 48 h and 1 week post-reperfusion by quantifying cell survival and assaying the integrity of myelin processes. There was progressive loss of GFP+ oligodendrocytes in ischemic white matter at 24 and 48 h. Surviving GFP+ cells had non-pyknotic nuclear morphology and were terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-negative, but there was marked fragmentation of myelin processes as early as 24 h after stroke. One week after stroke, we observed a restoration of GFP+ oligodendrocytes in ischemic white matter, reflected both by cell counts and by structural integrity of myelin processes. Proliferating cells were not the main source of GFP+ oligodendrocytes, as revealed by bromodeoxyuridine (BrdU) incorporation. These observations identify novel transient structural changes in oligodendrocyte cell bodies and myelinating processes, which may have consequences for white matter function after stroke.

Ultrastructural characterization of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced cell death in embryonic dopaminergic neurons.

Developing neuronal populations undergo significant attrition by natural cell death. Dopaminergic neurons in the substantia nigra pars compacta undergo apoptosis during synaptogenesis. Following this time window, destruction of the anatomic target of dopaminergic neurons results in dopaminergic cell death but the morphology is no longer apoptotic. We describe ultrastructural changes that appear unique to dying embryonic dopaminergic neurons. In primary cultures of mesencephalon, death of dopaminergic neurons is triggered by activation of glutamate receptors sensitive to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and differs ultrastructurally from both neuronal apoptosis or typical excitotoxicity. AMPA causes morphological changes selectively in dopaminergic neurons, without affecting other neurons in the same culture dishes. Two hours after the onset of treatment swelling of Golgi complexes is apparent. At 3 h, dopaminergic neurons display loss of membrane asymmetry (coinciding with commitment to die), as well as nuclear membrane invagination, irregular aggregation of chromatin, and mitochondrial swelling. Nuclear changes continue to worsen until loss of cytoplasmic structures and cell death begins to occur after 12 h. These changes are different from those described in neurons undergoing either apoptosis or excitotoxic death, but are similar to ultrastructural changes observed in spontaneous death of dopaminergic neurons in the natural mutant weaver mouse.

NMDA receptors mediate hypoxic spine loss in cultured neurons.

We examined the pharmacology of dendritic morphologic changes in cultured cortical neurons exposed to sublethal oxygen-glucose deprivation (OGD). Confocal analysis of DiI-labeled neurons demonstrated transient dendritic swelling and spine loss after OGD. These morphological changes were reproduced by direct application of NMDA, kainate, veratridine, ionomycyin, and gramicidin, but not KCl. Blockade of voltage-gated sodium or calcium channels did not prevent OGD-induced dendritic spine loss. In contrast, the NMDA receptor antagonist, MK-801, fully prevented these changes. An AMPA/kainate receptor antagonist, NBQX, had no effect by itself but reduced spine loss when added to MK-801. While alterations in dendrite morphology may be triggered by activation of disparate ion channels, rapid spine loss in hypoxic cortical neurons is mediated preferentially through activation of the NMDA subtype glutamate receptor.

Ampa/kainate receptor activation mediates hypoxic oligodendrocyte death and axonal injury in cerebral white matter.

We developed an in situ model to investigate the hypothesis that AMPA/kainate (AMPA/KA) receptor activation contributes to hypoxic-ischemic white matter injury in the adult brain. Acute coronal brain slices, including corpus callosum, were prepared from adult mice. After exposure to transient oxygen and glucose deprivation (OGD), white matter injury was assessed by electrophysiology and immunofluorescence for oligodendrocytes and axonal neurofilaments. White matter cellular components and the stimulus-evoked compound action potential (CAP) remained stable for 12 hr after preparation. OGD for 30 min resulted in an irreversible loss of the CAP as well as structural disruption of axons and subsequent loss of neurofilament immunofluorescence. OGD also caused widespread oligodendrocyte death, demonstrated by the loss of APC labeling and the gain of pyknotic nuclear morphology and propidium iodide labeling. Blockade of AMPA/KA receptors with 30 microm NBQX or the AMPA-selective antagonist 30 microm GYKI 52466 prevented OGD-induced oligodendrocyte death. Oligodendrocytes also were preserved by the removal of Ca(2+), but not by a blockade of voltage-gated Na(+) channels. The protective action of NBQX was still present in isolated corpus callosum slices. CAP areas and axonal structure were preserved by Ca(2+) removal and partially protected by a blockade of voltage-gated Na(+) channels. NBQX prevented OGD-induced CAP loss and preserved axonal structure. These observations highlight convergent pathways leading to hypoxic-ischemic damage of cerebral white matter. In accordance with previous suggestions, the activation of voltage-gated Na(+) channels contributes to axonal damage. Overactivation of glial AMPA/KA receptors leads to oligodendrocyte death and also plays an important role in structural and functional disruption of axons.

Dendritic spines lost during glutamate receptor activation reemerge at original sites of synaptic contact.

During cerebral ischemia, neurons undergo rapid alterations in dendritic structure consisting of focal swelling and spine loss. We used time-lapse microscopy to determine the fate of dendritic spines that disappeared after brief, sublethal hypoxic or excitotoxic exposures. Dendrite and spine morphology were assessed in cultured cortical neurons expressing yellow fluorescent protein or labeled with the fluorescent membrane tracer, DiI. Neurons exposed to NMDA, kainate, or oxygen-glucose deprivation underwent segmental dendritic beading and loss of approximately one-half of dendritic spines. Most spine loss was observed in regions of local dendritic swelling. Despite widespread loss, spines recovered within 2 hr after termination of agonist exposure or oxygen-glucose deprivation and remained stable over the subsequent 24 hr. Recovery was slower after NMDA than AMPA/kainate receptor activation. Time-lapse fluorescence imaging showed that the vast majority of spines reemerged in the same location from which they disappeared. In addition to spine recovery, elaboration of dendritic filopodia was observed in new locations along the dendritic shaft after dendrite recovery. Spine recovery did not depend on actin polymerization because it was not blocked by application of latrunculin-A, which eliminated filamentous actin staining in spines and blocked spine motility. Throughout spine loss and recovery, presynaptic and postsynaptic elements remained in physical proximity. These results suggest that elimination of dendritic spines is not necessarily associated with loss of synaptic contacts. Rapid reestablishment of dendritic spine synapses in surviving neurons may be a substrate for functional recovery after transient cerebral ischemia.

Amyloid-beta peptides are cytotoxic to oligodendrocytes.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive dementia. Amyloid-beta peptide (Abeta), a 39-43 amino acid peptide derived from beta-amyloid precursor protein, forms insoluble fibrillar aggregates that have been linked to neuronal and vascular degeneration in AD and cerebral amyloid angiopathy. Here we demonstrate that Abeta 1-40 and a truncated fragment, Abeta 25-35, induced death of oligodendrocytes (OLGs) in vitro in a dose-dependent manner with similar potencies. Abeta-induced OLG death was accompanied by nuclear DNA fragmentation, mitochondrial dysfunction, and cytoskeletal disintegration. Abeta activation of redox-sensitive transcription factors NF-kappaB and AP-1 and antioxidant prevention of Abeta-mediated OLG death suggest that oxidative injury contributes to Abeta cytotoxicity in OLGs. Recent demonstration of Abeta deposition and white matter abnormalities in AD implies a potential pathophysiological role for Abeta-mediated cytotoxicity of OLGs in this neurodegenerative disease.

Covalent and noncovalent interactions mediate metabotropic glutamate receptor mGlu5 dimerization.

Some, perhaps all, G protein-coupled receptors form homo- or heterodimers. We have shown that metabotropic glutamate receptors are covalent dimers, held together by one or more disulfide bonds near the N terminus. Here we report how mutating cysteines in this region affect dimerization and function. Covalent dimerization is preserved when cysteines 57, 93, or 99 are mutated but lost with replacement at 129. Coimmunoprecipitation under nondenaturing conditions indicates that the C[129]S mutant receptor remains a dimer, via noncovalent interactions. Both C[93]S and C[129]S bind [3H]quisqualate, whereas binding to C[57]S or C[99]S mutants is absent or greatly attenuated. The C[93]S and C[129]S receptors have activity similar to wild-type when assayed by fura-2 imaging of intracellular calcium in human embryonic kidney cells or electrophysiologically in Xenopus laevis oocytes. In contrast, C[57]S or C[99]S are less active in both assays but do respond with higher glutamate concentrations in the oocyte assay. These results demonstrate that 1) covalent dimerization is not critical for mGlu5 binding or function; 2) mGlu5 remains a noncovalent dimer even in the absence of covalent dimerization; and 3) high-affinity binding requires Cys-57 and Cys-99.

Oxygen-glucose deprivation induces inducible nitric oxide synthase and nitrotyrosine expression in cerebral endothelial cells.

BACKGROUND AND PURPOSE: The cerebral endothelial cells (ECs) are a primary target of hypoxic or ischemic brain insults. EC damage may contribute to postischemic secondary injury. Massive production of NO after inducible NO synthase (iNOS) expression has been implicated in cell death. This study aimed to characterize bovine cerebral EC death in relation to iNOS expression after oxygen-glucose deprivation (OGD) in vitro. METHODS: OGD in bovine cerebral ECs in culture was induced by deleting glucose in the medium and by incubating the cells in a temperature-controlled anaerobic chamber. The extent of cell death was assessed by trypan blue exclusion, MTT assay, and LDH release. ELISA, gel electrophoresis, and staining by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling were used to examine DNA fragmentation. The expression of iNOS mRNA and protein was detected by reverse transcription-polymerase chain reaction and Western blotting, respectively. Nitrotyrosine expression was confirmed with Western blot analysis and immunostaining. RESULTS: Bovine cerebral EC death was dependent on the duration of OGD and showed selected biochemical, morphological, and pharmacological features suggestive of apoptosis. OGD also induced the expression of iNOS mRNA and protein in bovine cerebral ECs. Increased expression of nitrotyrosine, the product formed by peroxynitrite reaction with proteins, was also detected after OGD. The involvement of iNOS in EC death was suggested by partial reduction of cell death by NO synthase inhibitors, including L-N(G)-(1-iminoethyl)ornithine and nitro-L-arginine, and an NO scavenger, the Fe(2+)-N-methyl-D-glucamine dithiocarbamate complex. CONCLUSIONS: OGD-induced bovine cerebral EC death involves an apoptotic process. Induction of iNOS with subsequent peroxynitrite formation may contribute to bovine cerebral EC death caused by OGD.

Ionic selectivity of low-affinity ratiometric calcium indicators: mag-Fura-2, Fura-2FF and BTC.

Accurate measurement of elevated intracellular calcium levels requires indicators with low calcium affinity and high selectivity. We examined fluorescence spectral properties and ionic specificity of three low-affinity, ratiometric indicators structurally related to Fura-2: mag-Fura-2 (furaptra), Fura-2FF, and BTC. The indicators differed in respect to their excitation wavelengths, affinity for Ca2+ (Kd approximately 20 microM, 6 microM and 12 microM respectively) and selectivity over Mg2+ (Kd approximately 2 mM for mag-Fura-2, > 10 mM for Fura-2FF and BTC). Among the tested indicators, BTC was limited by a modest dynamic range upon Ca2+ binding, susceptibility to photodamage, and sensitivity to alterations in pH. All three indicators bound other metal ions including Zn2+, Cd2+ and Gd3+. Interestingly, only in the case of BTC were spectral differences apparent between Ca2+ and other metal ions. For example, the presence of Zn2+ increased BTC fluorescence 6-fold at the Ca2+ isosbestic point, suggesting that this dye may be used as a fluorescent Zn2+ indicator. Fura-2FF has high specificity, wide dynamic range, and low pH sensitivity, and is an optimal low-affinity Ca2+ indicator for most imaging applications. BTC may be useful if experimental conditions require visible wavelength excitation or sensitivity to other metal ions including Zn2+.

Expression of the tuberous sclerosis complex gene products, hamartin and tuberin, in central nervous system tissues.

Tuberous sclerosis complex (TSC) is a common genetic disorder in which affected individuals can develop mental retardation, developmental brain defects, and seizures. Two genetic loci are responsible for TSC: TSC1 on chromosome 9q and TSC2 on chromosome 16p. Here, we report our analysis of TSC1 (hamartin) and TSC2 (tuberin) protein expression in the central nervous system (CNS). Both tuberin and hamartin are expressed in neurons and astrocytes where they physically interact. In the mouse cerebellum in vivo, tuberin predominantly localizes to the perinuclear region of the Purkinje cell, whereas hamartin is distributed along neuronal or astrocytic processes. In contrast, both hamartin and tuberin demonstrate similar neuronal expression patterns in pure neuronal cultures in vitro. Additionally, hamartin is highly expressed in astrocytes in mixed neuron-glia cultures in vitro, suggesting that hamartin may be important for astrocyte growth control. Unlike tuberin, loss of hamartin expression was not observed in sporadic astrocytomas. These results suggest that tuberin and hamartin may differentially contribute to the CNS pathology in TSC.

Calcium ionophores can induce either apoptosis or necrosis in cultured cortical neurons.

Cultured cortical neurons exposed for 24 h to low concentrations of the Ca2+ ionophores, ionomycin (250 nM) or A-23187 (100 nM), underwent apoptosis, accompanied by early degeneration of neurites, cell body shrinkage, chromatin condensation and internucleosomal DNA fragmentation. This death could be blocked by protein synthesis inhibitors, as well as by the growth factors brain-derived neurotrophic factor or insulin-like growth factor I. If the ionomycin concentration was increased to 1-3 microM, then neurons underwent necrosis, accompanied by early cell body swelling without DNA laddering, or sensitivity to cycloheximide or growth factors. Calcium imaging with Fura-2 suggested a possible basis for the differential effects of low and high concentrations of ionomycin. At low concentrations, ionomycin induced greater increases in intracellular Ca2+ concentration in neurites than in neuronal cell bodies, whereas at high concentrations, ionomycin produced large increases in intracellular Ca2+ concentration in both neurites and cell bodies. We hypothesize that the ability of low concentrations of Ca2+ ionophores to raise intracellular Ca2+ concentration preferentially in neurites caused early neurite degeneration, leading to loss of growth factor availability to the cell body and consequent apoptosis, whereas high concentrations of ionophores produced global cellular Ca2+ overload and consequent necrosis.

Oligodendrocytes from forebrain are highly vulnerable to AMPA/kainate receptor-mediated excitotoxicity.

Little is known of the molecular mechanisms that trigger oligodendrocyte death and demyelination in many acute central nervous system insults. Since oligodendrocytes express functional alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate-type glutamate receptors, we examined the possibility that oligodendrocyte death can be mediated by glutamate receptor overactivation. Oligodendrocytes in primary cultures from mouse forebrain were selectively killed by low concentrations of AMPA, kainate or glutamate, or by deprivation of oxygen and glucose. This toxicity could be blocked by the AMPA/kainate receptor antagonist 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2,3-dione (NBQX). In vivo, differentiated oligodendrocytes in subcortical white matter expressed AMPA receptors and were selectively injured by microstereotaxic injection of AMPA but not NMDA. These data suggest that oligodendrocytes share with neurons a high vulnerability to AMPA/kainate receptor-mediated death, a mechanism that may contribute to white matter injury in CNS disease.

Distinct roles for sodium, chloride, and calcium in excitotoxic dendritic injury and recovery.

The postsynaptic neuronal dendrite is selectively vulnerable to hypoxic-ischemic brain injury and glutamate receptor overactivation. We explored the glutamate receptor pharmacology and ionic basis of rapid, reversible alterations in dendritic shape which occur in cultured neurons exposed to glutamate. Dendrite morphology was assessed with the fluorescent membrane tracer, DiI, or immunofluorescence labeling of the somatodendritic protein, MAP2. Cortical cultures derived from 15-day-old mouse embryos underwent segmental dendritic beading when exposed to NMDA, AMPA, or kainate, but not to metabotropic glutamate receptor agonists. Varicosity formation in response to NMDA or kainate application was substantially attenuated in reduced sodium buffer (substituted with N-methyl-D-glucamine). Furthermore, veratridine-induced sodium entry mimicked excitotoxic alterations in dendrites and additionally caused varicosity formation in axons. Solutions deficient in chloride (substituted with Na methylsulfate) and antagonists of chloride-permeable GABA/glycine receptors reduced NMDA- or kainate-induced varicosity formation. An increase in dendrite volume was observed as varicosities formed, and varicosity formation was attenuated in sucrose-supplemented hypertonic media. Despite marked structural changes affecting virtually all neurons, dendrite shape returned to normal within 2 h of terminating glutamate receptor agonist application. Neurons exposed to kainate recovered more rapidly than those exposed to NMDA, and neurons exposed to NMDA in calcium-free buffer recovered more rapidly than cells treated with NMDA in normal buffer. While sodium, chloride, and water entry contribute to excitotoxic dendritic injury acutely, calcium entry through NMDA receptors results in lasting structural changes in damaged dendrites.

Neuronal free calcium measurement using BTC/AM, a low affinity calcium indicator.

BTC is a low affinity calcium indicator (Kd approximately 7-26 microM) featuring many desirable properties for cellular calcium imaging, including long excitation wavelengths (400/485 nm), low sensitivity to Mg2+, and accuracy of ratiometric measurement [Iatridou H., Foukaraki E., Kuhn M.A., Marcus E.M., Haugland R.P., Katerinopoulos H.E. The development of a new family of intracellular calcium probes. Cell Calcium 1994; 15: 190-198]. To assess the usefulness of this indicator in cultured neurons, we examined properties of BTC and its acetoxymethyl ester, BTC/AM. BTC/AM had substantial calcium-independent fluorescence at all excitation wavelengths. BTC/AM was readily loaded into neurons and was rapidly hydrolysed. There was little dye compartmentalization, as assessed by digitonin lysis, Co2+ quenching of BTC fluorescence and by confocal microscopy. Despite adequate loading, BTC gradually became unresponsive to [Ca2+]i when cultures were examined under routine imaging conditions. This effect was a function of the cumulative fluorescence illumination and could be minimized by attenuating light intensity or duration. Ratio imaging after exposure of neuronal cultures to 1-50 microM ionomycin revealed distinct sensitivity ranges for BTC and Fura-2. BTC reported graded neuronal [Ca2+]i responses to glutamate receptor stimulation with N-methyl-D-aspartate in the range 10-50 microM, whereas Fura-2 did not distinguish between these stimuli. Under appropriate loading and illumination conditions, bath-loaded BTC/AM may be well suited for measurement of moderate to high calcium concentrations in cultured neurons.

Ionized intracellular calcium concentration predicts excitotoxic neuronal death: observations with low-affinity fluorescent calcium indicators.

Cytosolic calcium ([Ca2+]i) is an important mediator of neuronal signal transduction, participating in diverse biochemical reactions that elicit changes in synaptic efficacy, metabolic rate, and gene transcription. Excessive [Ca2+]i also has been implicated as a cause of acute neuronal injury, although measurement of [Ca2+]i in living neurons by fluorescent calcium indicators has not consistently demonstrated a correlation between [Ca2+]i and the likelihood of neuronal death after a variety of potentially lethal insults. Using fluorescence videomicroscopy and microinjected calcium indicators, we measured [Ca2+]i in cultured cortical neurons during intense activation with either NMDA (300 microM) or AMPA (450 microM). At these concentrations NMDA killed >80% of the cultured neurons by the next day, whereas neuronal death from AMPA was <20%. Using the conventional calcium indicator, fura-2/AM, we estimated [Ca2+]i elevations to be approximately 300-400 nM during exposure to either glutamate agonist. In contrast, indicators with lower affinity for calcium, benzothiazole coumarin (BTC), and fura-2/dextran reported [Ca2+]i levels >5 microM during lethal NMDA exposure, but [Ca2+]i levels were <1.5 microM during nonlethal activation of AMPA receptors or voltage-gated calcium channels. Fura-2 reported [Ca2+]i responses during brief exposure to glutamate, NMDA, AMPA, kainate, and elevated extracellular K+ between 0.5 and 1 microM. With the use of BTC, only NMDA and glutamate exposures resulted in micromolar [Ca2+]i levels. Neurotoxic glutamate receptor activation is associated with sustained, micromolar [Ca2+]i elevation. The widely used calcium indicator fura-2 selectively underestimates [Ca2+]i, depending on the route of entry, even at levels that appear to be within its range of detection.

Calpain activation contributes to dendritic remodeling after brief excitotoxic injury in vitro.

The calcium-dependent protease calpain may contribute to neuronal death in acute neurological insults and may be activated very early in the neuronal injury cascade. We assessed the role of calpain in a model of rapid, reversible dendritic injury in murine cortical cultures. Brief sublethal NMDA exposure (10-30 microM for 10 min) resulted in focal swellings, or varicosities, along the length of neuronal dendrites as visualized with the lipophilic membrane tracer Dil or with immunostaining using antibodies to the somatodendritic protein MAP2. These varicosities appeared within minutes of NMDA exposure and recovered spontaneously within 2 hr after NMDA removal. Addition of the calpain inhibitors MDL28,170, calpain inhibitors I and II, and leupeptin (all 1-100 microM) had little effect on the development of NMDA-induced dendrite injury. However, the resolution of varicosities was substantially delayed by addition of calpain inhibitors after sublethal excitotoxic exposure. Using Western blots and immunocytochemistry, we observed reactivity for a calpain-specific spectrin proteolytic fragment during the period of recovery from dendritic swelling, but not during its formation. Spectrin breakdown product immunoreactivity could be blocked by the calpain inhibitor MDL28,170 and appeared in neuronal cell bodies and neurites in a time course that paralleled dendritic recovery. These observations suggest that calcium-dependent proteolysis contributes to recovery of dendritic structure after NMDA exposure. Calpain activation is not necessarily detrimental and may play a role in dendritic remodeling after neuronal injury.

Cyclosporine induces neuronal apoptosis and selective oligodendrocyte death in cortical cultures.

Cyclosporine is used clinically as an immunosuppressant, but carries a risk of central nervous system toxicity due to undefined mechanisms. We examined the ability of cyclosporine exposure to kill cultured mouse cortical neurons and glia. Mixed neuron/glial cultures exposed to 1 to 20 microM cyclosporine for 24 to 48 hours developed concentration-dependent neuronal death, with most neurons destroyed by 20 microM cyclosporine. This neuronal death was characterized by cell body shrinkage and blebbing, chromatin condensation, and internucleosomal DNA fragmentation, consistent with apoptosis. Neuronal death was reduced by addition of cycloheximide, brain-derived neurotrophic factor, or insulin-like growth factor I but not N-methyl-D-aspartate- or AMPA-type glutamate receptor antagonists. Oligodendrocytes were more sensitive to cyclosporine-induced damage than were neurons, but astrocytes were relatively resistant. Oligodendrocyte death was accompanied by positive TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling) staining and was attenuated by application of ciliary neurotrophic factor or insulin-like growth factor I but not glutamate receptor antagonists. Present observations raise the possibility that the central nervous system toxicity syndrome associated with cyclosporine may be caused by the drug-induced death of oligodendrocytes and neurons.

Postpartum cerebral vasospasm treated with hypervolemic therapy.

In a 23-year-old woman, gravida 1, para 1-0-0-1, headaches and seizures developed 1 week after an uncomplicated delivery. Cerebral angiography revealed severe, diffuse cerebral vasospasm. Her symptoms resolved with hyperosmolar, hypervolemic therapy and nimodipine. Magnetic resonance angiography on postpartum day 23 confirmed persistent, severe vasospasm, and repeat magnetic resonance angiography on postpartum day 33 demonstrated interval improvement. This report documents the time course of a case of postpartum vasospasm and its response to hypervolemic, hyperosmolar therapy and nimodipine.

Automated measurement of infarct size with scanned images of triphenyltetrazolium chloride-stained rat brains.

BACKGROUND AND PURPOSE: The extent of brain infarction after local cerebral ischemia is frequently assessed with the mitochondrial activity indicator 2,3,5-triphenyltetrazolium chloride (TTC). We describe an automated procedure for analysis of infarct size in TTC-stained rat brains. METHODS: Rats were subjected to middle cerebral artery occlusion and killed after 24 to 36 hours, and their brains were processed for TTC staining. Digital images of coronal sections from these brains (n > 50) were acquired with a desktop color scanner. The resulting images were divided into red, blue, and green component images. Total brain and infarct areas were automatically determined on the basis of total pixel intensity and area after segmentation of the red and green images, respectively. Automated measurements were compared with those made with a video camera-based image acquisition system that required manual tracing of lesion boundaries. RESULTS: The spatial resolution of scanned brain images (approximately equal to 200 microns) was comparable to that of the camera-based system and provided sufficient detail to recognize infarct boundaries and neuroanatomical features. Scanner-based acquisition and analysis were faster than with the camera-based method. The green component image accurately distinguished infarcted from normal brain, and the red component image represented total brain dimensions. Infarct measurements obtained by the automated method correlated closely with those from conventional apparatus (R2 = .89, P < .001). Intraobserver reliability with the automated method (R2 = 1.00) was higher than with the conventional method (R2 = .77). CONCLUSIONS: Infarct size after middle cerebral artery occlusion in the rat can be rapidly and reproducibly assessed with inexpensive scanning equipment and automated image analysis of TTC-stained brains.