Rapid quantification of ischaemic injury and cerebroprotection in brain slices using densitometric assessment of 2,3,5-triphenyltetrazolium chloride staining

2,3,5-Triphenyltetrazolium chloride (TTC), a marker of mitochondrial enzyme activity, is widely used to assess the effects of cerebral ischaemia in vivo. In the present study, we characterised its utility as a simple rapid macrohistological measure of ischaemic damage in brain slices. Coronal rat corticostriatal slices were incubated in oxygenated artificial cerebrospinal fluid (aCSF) until subjected to 'ischaemia' (deoxygenated, hypoglycaemic aCSF) for up to 12 min. After a further 30 min to 16 h of reincubation in oxygenated aCSF, slices were stained with TTC, fixed with formalin and transferred to cover slips. The slices were scanned in 8-bit greyscale using a standard desktop scanner and the staining analysed by densitometry of the acquired images. Control slices stained a rich pink/red. Ischaemia (10 min) reduced both the area and intensity of staining. Both measures of striatal staining were negatively correlated with the duration of ischaemia (0-12 min). Furthermore, staining in the striatum correlated significantly with cortical TTC staining. The effects of TTC concentration (0.063-0.5% w/v) and post-ischaemic interval (30 min to 16 h) were examined upon the intensity of TTC staining. (+)-MK 801 prevented the ischaemia-induced reduction in TTC staining, consistent with cerebroprotection. These data suggest that TTC staining of brain slices may be used to quantify ischaemic injury and cerebroprotection.     

Comparative study of 2,3,5-triphenyltetrazolium chloride (TTC) and hematoxylin-eosin staining for quantification of early brain ischemic injury in cats

There are no staining methods that can reliably and unequivocally detect final infarcts in the acute stage of experimental ischemia. In most instances, 2,3,5-triphenyltetrazolium chloride (TTC) and hematoxylin and eosin (HE) stainings are accepted for this purpose, but neither is perfect. We performed a comparative study of the TTC immersion method and HE staining for quantification of early brain ischemic injury in cats, focussing on the reproducibility associated with planimetry. Focal brain ischemia was produced by middle cerebral artery (MCA) occlusion via the transorbital approach in 14 cats. After 6 h of occlusion, two slices of 3 mm thickness, passing through the optic chiasma and mammillary body, were selected for pathological examination. TTC immersion and HE staining were both used for planimetric study of the surface of the same slice. The area of the injury was traced manually with the aid of computerized digital planimetry and expressed as a percentage of the area of the contralateral hemisphere. The area of hemispheric injury and the area of gray matter injury were separately calculated in the TTC specimens, and each was compared with the area of gray matter injury in the HE-stained specimens. Measurements were repeated twice on each slice to estimate errors associated with manual tracing of the boundary of the area of injury. The mean percentage values of the area of injury in the TTC-immersed gray matter specimens were lower than those detected by HE staining, although there was a very significant correlation between the two. The differences between each two measurements of TTC-determined gray matter injury were significantly less than those between each two measurements of HE-determined gray matter injury. The differences between each two measurements of TTC-determined hemispheric injury were slightly less than those between each two measurements of TTC-determined gray matter injury, although there was no significant difference between them. For quantifying ischemic injury after 6 h of MCA occlusion in cats, the TTC immersion method is more reproducible and simpler in manner than HE staining, but the results of both are significantly correlated.     

Evaluation of an optimal temperature for brain storage in delayed 2, 3,5-triphenyltetrazolium chloride staining

This paper presents the determination of an optimal temperature for delayed 2,3,5-triphenyltetrazolium chloride (TTC) staining. Twenty-one rats were subjected to right middle cerebral artery embolic stroke and sacrificed 96 h following ischemia. The brains were harvested and stained immediately after sacrifice or stored for 8 h at 21-23 degrees C or 4 degrees C, respectively. The stained sections were scanned and infarct volume calculated. The quality of staining, distinction of borders between infarcted and non-ischemic tissue and ease of differentiating ischemic tissue in colored and grayscale images was assessed. The present study indicates that results of TTC obtained immediately after animal sacrifice, or delayed TTC staining while storing the brains at room temperature or 4 degrees C are comparable.     

Mannitol-facilitated perfusion staining with 2,3,5-triphenyltetrazolium chloride (TTC) for detection of experimental cerebral infarction and biochemical analysis

A simple method to quantify cerebral infarction has great value for mechanistic and therapeutic studies in experimental stroke research. Immersion staining of unfixed brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) is a popular method to determine cerebral infarction in preclinical studies. However, it is often difficult to apply immersion TTC-labeling to severely injured or soft newborn brains in rodents. Here we report an in vivo TTC perfusion-labeling method based on osmotic opening of blood-brain-barrier with mannitol-pretreatment. This new method delineates cortical infarction correlated with the boundary of morphological cell injury, differentiates the induction or subcellular redistribution of apoptosis-related factors between viable and damaged areas, and easily determines the size of cerebral infarction in both adult and newborn mice. Using this method, we confirmed that administration of lipopolysaccharide 72 h before hypoxia-ischemia increases the damage in neonatal mouse brains, in contrast to its effect of protective preconditioning in adults. These results demonstrate a fast and inexpensive method that simplifies the task of quantifying cerebral infarction in small or severely injured brains and assists biochemical analysis of experimental cerebral ischemia.     

Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats

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.     

A reversible component of cerebral injury as identified by the histochemical stain 2,3,5-triphenyltetrazolium chloride (TTC)

Summary The extent of histochemical change following middle cerebral artery occlusion was quantitatively determined in three groups of Sprague-Dawley rats with 2,3,5-triphenyltetrazolium chloride (a marker of mitochondrial oxidative enzyme function). In group I (n=7) occlusion was maintained for 3 h, with immediate sacrifice. In group II (n=7) occlusion was maintained for 5 h, with immediate sacrifice. In group III (n=7) occlusion was maintained for 3 h, followed by a 2-h period of reperfusion prior to sacrifice. The area of injury was significantly larger (P<0.05) in the 5-h occlusion group [15±4% (mean±SD)] compared to the 3-h occlusion group (9±2%); indicating a time-dependent worsening of the histochemical detection of injury. However, the area of injury was significantly less in the reperfusion group (5±2%) compared to the group that was evaluated after 3 h of occlusion without reperfusion (9±2%); indicating that some component of the injury revealed by 2,3,5-triphenyltetrazolium chloride is potentially reversible. These data suggest that contrary to previous understanding, the histochemical abnormality revealed by 2,3,5-triphenyltetrazolium chloride is reversible in some circumstances and does not necessarily represent inevitable infarction.Supported in part by a Research Starter Grant from the American Society of Anesthesiologists     

Hypoglycemic coma associated with brain infarcts.

Hypoglycemic hemiplegia may lead to a mistaken diagnosis of stroke, although the symptoms resolve with correction of the hypoglycemia. We report a 27-year-old white man with insulin-dependent diabetes who developed right hemispheric infarcts and left hemiplegia associated with hypoglycemic coma. This report discusses the possible role of hypoglycemia in causing the stroke.     

Development of transient acetylcholinesterase staining in cells and permanent staining in fibers in cortex of rat brain.

The development of the acetylcholinesterase (AChE) texture of the cortex of the rat brain was studied during the first three weeks of life. The Tago technique enables visualization of both AChE+ cells and fibers with both shown in exquisite detail making quantification possible. At each age--0 (birth), 7, 14, 21 and 60 days (adult)--four brain areas were studied (cingulate, dorsal neocortex, lateral neocortex and olfactory) at each of three coronal planes in the brain (anterior, intermediate, posterior). Fiber density reached adult levels by Day 21 in cingulate cortex in intermediate and posterior planes. In other areas fiber density reached adult levels by Day 14 indicating a high rate of fiber growth during the first two weeks of life since at birth rat cortex is innervated only by a sparse AChE+ fiber invasion into neocortex in the anterior plane. Fiber density did not regress after adult levels were reached, however, cell staining showed a different pattern. At birth many lightly stained cells were seen in the olfactory cortex in all three planes, but other areas were devoid of cells. In all areas there was a peak at Day 7 in number of cells stained and in intensity of cells staining with a gradual decline in cell staining until by Day 21 very few stained cells were seen in the cortex (typical adult pattern).     

Cogan''s syndrome complicated by lacunar brain infarcts.

Cogan's syndrome, nonsyphilitic interstitial keratitis with vestibuloauditory dysfunction, is an uncommon disease of young adults, probably a manifestation of vasculitis. A 32 year old woman with this syndrome developed a thalamic syndrome with amnesia and dysphasia due to lacunar infarcts.     

Aphasia owing to subcortical brain infarcts in childhood

The aim of this study was to further define the clinical features of subcortical aphasia in children with deep brain infarcts and to define the sequelae associated with childhood strokes. We retrospectively studied nine children with left subcortical brain infarcts who presented with acquired language disorder and underwent language investigations based on standardized tests. Stroke in these patients involved the left internal capsule, lenticular or thalamic nuclei, or a combination of these. Early aphasic manifestations following the deep cerebral infarcts affected language expression. These included mutism, nonfluent speech, word finding difficulties, and phonemic and semantic paraphasia. Speech comprehension was generally more preserved. All patients subsequently improved, although variably; sequelae such as dysfluency, word finding difficulties, and written language learning impairment could be detected through standardized tests in six of them (all younger than 6 years at the time of the infarct). Two of the three remaining patients (both older than 6 years at the time of the infarct) had a full recovery. Our study confirms the concept of childhood subcortical aphasia, depicts the linguistic profile in these patients, and sustains the indication of systematic formal language assessment during the follow-up of all children with subcortical infarct involving the dominant hemisphere.     

Cerebrovascular permeability and brain edema after cortical photochemical infarcts in the rat

The importance of protein extravasation for the development of vasogenic brain edema is still controversial. We, therefore, assessed the cerebrovascular permeability to serum proteins in relation to the development and resolution of brain edema in a photochemical cortical lesion in the rat. Cortical infarction was induced by in situ thrombosis using an argon laser beam aimed at the exposed parietal bone in animals given rose bengal i.v. The histology and the cerebrovascular permeability to serum proteins were scrutinized from 2 h to 3 weeks after the insult. The presence of serum proteins was demonstrated by an immunoperoxidase technique. The cerebral water content was estimated by specific gravity measurements of the cortical tissue in a kerosene-monobromobenzene gradient column from 2 h to 7 days after infarction. The blood-brain barrier was permeable to proteins at 2 h following the insult and proteins spread into the medial and lateral tissue reaching a maximum at 24 h. The specific gravity did not deviate from control values at 2 h. After 8 h the specific gravity of the lesion decreased with smaller decreases in the immediately afjacent tissue. At 24 h the changes in specific gravities reached a maximum in all regions except the immediately lateral area. The edema was generally worse in tissue medial to rather than lateral to the infarct. The degradation of serum proteins and the resolution of the brain edema followed the same time course with partial resolution of 72 h. By 1 week serum proteins and edema were confined to the central necrotic core. The results suggest a relationship between cerebrovascular permeability and cerebral edema in photochemical cortical infarction.     

Use of reduced silver staining to show loss of connections in aged rat brain

A variation of the Fink-Heimer reduced silver technique was used to show the extent of axonal degeneration in the brains of aged rats. Degeneration product was extensive throughout the white matter of the old brains, being most dense in the optic tract, but substantial in fornix, corpus callosum, cingulum, and anterior commissure. There was selective impregnation of hippocampal strata, suggesting that there may be differential atrophy of connections as a function of age.     

C-fos expression in the rat brain after intraperitoneal injection of lithium chloride.

The distribution of evoked expression of the proto-oncogene c-fos was immunohistochemically examined in the rat brain after intraperitoneal injection of isotonic LiCl, which is commonly used to induce internal malaise in the conditioned taste aversion paradigm. C-fos-like immunoreactive neurones (c-fos neurones) were most densely observed in the central amygdaloid nucleus, external lateral subnucleus of the parabrachial nucleus (PBN), posteromedial and commissural parts of the nucleus of the tractus solitarius (NTS) and area postrema (AP). Experiments including vagotomy, intravenous injection of LiCl and lesions of the area postrema suggest that NTS neurones are activated via both sides of the vagus nerves, while AP neurones, humorally as well as neurally via the vagal nerve with a right side predominance. The activated NTS and AP neurones project mainly to the external lateral subnucleus of the PBN and lightly to the central lateral subnucleus of the PBN. These results are discussed in terms of the role of LiCl in the formation of conditioned taste aversion.     

Characterization of barbiturate-stimulated chloride efflux from rat brain synaptoneurosomes

Membrane chloride (Cl-) permeability was studied in a novel subcellular brain preparation, the synaptoneurosome. Using a radioactive tracer exchange technique, Cl- transport was determined by measuring 36Cl- efflux from rat cerebral cortical synaptoneurosomes. Barbiturates increased 36Cl- efflux in a dose-dependent manner with the following relative order of potency: 5-(1,3-dimethylbutyl)-5-ethyl barbituric acid ((-)-DMBB) greater than pentobarbital greater than secobarbital greater than (+)-DMBB greater than hexobarbital greater than amobarbital greater than mephobarbital. Phenobarbital and barbital were virtually inactive. A good correlation was observed between the potencies of these barbiturates in stimulating 36Cl- efflux and their anesthetic potencies in mice (r = 0.90, p less than 0.01) and their abilities to enhance [3H] diazepam binding to brain membranes (r = 0.77, p less than 0.05). The effect of pentobarbital in enhancing 36Cl- efflux was reversed by the gamma-aminobutyric acid (GABA) antagonists picrotoxin and bicuculline. Picrotoxin and bicuculline both decreased 36Cl- efflux in the absence of pentobarbital, suggesting the presence of endogenous GABA. Incubation of synaptoneurosomes with 4,4'-di-isothiocyano- or dinitro-2,2'-disulfonic acid stilbene, inhibitors of anion transport, also decreased both basal and pentobarbital-induced 36Cl- efflux. Pentobarbital (500 microM) was most effective in inducing 36Cl- efflux in the cerebellum, hippocampus, and cortex (23.7, 23.6, and 22.5%, respectively), and was less effective in stimulating 36Cl- efflux in the striatum (15.1%) and pons-medulla (6.2%). The relative efficacy of pentobarbital in enhancing 36Cl- efflux among these various brain regions was highly correlated (r = 0.96, p 0.01) with the relative densities of [35S]-t-butylbicyclophosphorothionate-binding sites, a measure of GABA-gated Cl- channel density.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of glycogen phosphorylase a and cytochrome oxidase histochemical staining in rat brain.

The utility of metabolic markers that index functional neuronal circuits is widely appreciated. The present study asks whether patterns of the metabolic enzyme, active glycogen phosphorylase, parallel those of the neuronal marker, cytochrome oxidase. Fresh frozen rat brain sections (30 microns) were processed for either active glycogen phosphorylase or cytochrome oxidase at each of ten levels of the neuraxis. Although these metabolic markers predominate in different cellular compartments--glycogen phosphorylase in the astrocytic compartment and cytochrome oxidase in the neuronal compartment--the patterns of high, moderate, and low levels of activity for both enzymes were generally parallel. These similarities extended to detailed patterns of heterogeneous staining within structures, in particular, to laminated and modular distribution within cerebral and cerebellar cortical structures. The modular distribution was evident in barrel structures in the cerebral cortex and in parasagittal compartments in the vermis of the cerebellum. Conspicuous differences between the two patterns occurred in white matter, in subcortical grey matter regions such as the nucleus accumbens, diagonal band, amygdala, and globus pallidus, and in the superior olivary nuclei of the brainstem as well as in nonneural structures such as the choroid plexus and ependyma. Discrete patchiness was characteristic of active glycogen phosphorylase distribution in the limbic neuropil of the dentate gyrus and entorhinal cortex. The strong parallels between active glycogen phosphorylase and cytochrome oxidase distribution support the view that glycogen phosphorylase, despite its glial localization, can reflect neuronal metabolic demands.     

Ultrastructural alterations of brain cortex in rat following intraperitoneal administration of mercuric chloride.

Intraperitoneal administration of a single dose (6 mg/kg body weight) of mercuric chloride (HgCl2) results in ultrastructural changes in brain cortex of the rats. 18 hours after administration of HgCl2 the accumulation of dense deposits of mercury in nerve and glial cell cytoplasm was observed. The quantity of microglia in neuropil was also increased in this experimental group. We postulate that these cerebral macrophages can play an important role in the process of intoxication, too. 5 days after administration of mercuric chloride many of these changes reversed.     

An optimized triphenyltetrazolium chloride method for identification of cerebral infarcts

2,3,5-Triphenyltetrazolium chloride (TTC) staining is a convenient procedure for detection of brain infarcts but no standardized procedure is available. We report here an optimized and economic procedure of staining with TTC. Rats were subjected to reversible middle cerebral artery (MCA) occlusion (2-h ischemia and 24-h reperfusion). At the end of reperfusion, brain was isolated and sliced rostro-caudally into serial 2-mm-thick slices. Sets of three serial slices from each brain were incubated for 30 min at 37 degrees C in three different concentrations of TTC-the first slice of the set in 1%, the second in 0.05% and the third in 0.1% TTC-in phosphate-buffered saline. Staining characteristics, optical density (OD) and infarct size were compared between juxtaposing cut surfaces of the slices stained with the three concentrations of TTC. After the first use, 0.05% TTC solution was stored at 4-8 degrees C and reused on the same day or on subsequent days. TTC at 0.05% concentration provided high contrast staining with clear demarcation between normal and infarct tissue. The infarct size in 0.05% TTC-stained slices correlated well with that in 0.1% TTC (r=0.92)- and 1% TTC (r=0.93)-stained preparations. 'Nonspecific' staining of corpus callosum and the anterior commissures was minimal with the method. Once-used 0.05% TTC solution could be stored at 4-8 degrees C and reused. In conclusion, staining with 0.05% TTC provided improved delineation of brain infarcts, reduced 'nonspecific' staining of white matter and the infarct size correlated well with that measured after 1% TTC staining; the method also reduces the costs to 1/20.     

Multiple brain infarcts and Balint syndrome in aortic arch angiosarcoma.

A 50-year-old woman presented with subacute cognitive decline, impaired eye movements, and simultanagnosia, components of the Balint syndrome. She had relatively low blood pressure in the left arm and left finger clubbing. Brain imaging identified multiple acute infarcts. Transesophageal echocardiography showed no cardiac abnormalities but demonstrated a thickened aortic wall and an intraluminal aortic arch mass. The surgical specimen revealed angiosarcoma. Of the few reported angiosarcomas involving the aorta, most have been located in the abdominal segment. This is only the second reported case of aortic arch sarcoma presenting with stroke.