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Background and Purpose— Thrombin-induced clots used in experimental thromboembolic stroke differ from clots forming spontaneously under clinical conditions. We investigated whether this difference influences the efficacy and outcome of thrombolytic treatment. Methods— In rats, the middle cerebral artery was occluded by intracarotid injection of fibrin-rich clots, prepared either according to established methods by adding thrombin to freshly drawn arterial blood or by spontaneous coagulation. The mechanical properties of clots were determined in vitro by elasticity and plasticity tests. One hour after embolism, thrombolysis was started by intra-arterial application of recombinant tissue plasminogen activator (rtPA) (10 mg/kg). Treatment efficacy was monitored by MR measurements of blood perfusion, apparent diffusion coefficient (ADC), T2 relaxation time and blood-brain barrier permeability, and by pictorial measurements of ATP and pH. Results— Thrombin-induced clots were classified as elastic, and spontaneously forming clots were classified as plastic. Middle cerebral artery embolism with thrombin-induced or spontaneously forming clots led to similar reduction of perfusion and ADC, but rtPA treatment efficacy differed greatly. In the spontaneously forming clot group, blood perfusion returned to or above control within 2 hours, ADC and ATP normalized, tissue pH exhibited alkalosis, and T2 and blood-brain barrier permeability did not change. In the thrombin-induced clot group, in contrast, blood reperfusion was delayed, ADC and ATP remained reduced, tissue pH was acidic, and edema developed, as reflected by increased T2 and blood-brain barrier permeability. Conclusions— rtPA-induced thrombolysis promotes rapid reperfusion and tissue recovery in animals embolized with spontaneously forming clots but not in those embolized with thrombin-induced clots. This difference is explained by the different mechanical and possibly molecular consequences of clot preparation and must be considered for the interpretation of thrombolysis experiments.

Diffusion-weighted nuclear magnetic resonance imaging has been shown to detect early ischemia-related alterations in experimental stroke. This raises the question of whether the observed increase in signal intensity is correlated with changes in cerebral metabolism. After middle cerebral artery occlusion, nuclear magnetic resonance diffusion images were recorded and compared with the regional concentration of cerebral metabolites and with histology of identical planes. Seven anesthetized Fischer rats were subjected to permanent occlusion of the middle cerebral artery. T1, T2, and diffusion images (b factors ranging from 0 to 1500 s/mm2) were measured in three to five planes after 7 hours. Thereafter, brains were frozen in situ for histology and quantitative bioluminescence imaging of ATP, glucose, lactate, and for fluorescence imaging of tissue pH. Seven hours after middle cerebral artery occlusion, the apparent diffusion coefficient was reduced from 615 +/- 97 x 10(-6).mm2.s-1 (contralateral brain) to 359 +/- 42 x 10(-6).mm2.s-1 (ischemic brain; mean +/- SD, P < .01). A precise topical coincidence was demonstrated between changes in nuclear magnetic resonance diffusion images, pattern of histological damage, ATP-depleted areas, and local tissue acidosis, the lesion area amounting to between 24.1% and 27.6% of the hemisphere at the level of the caudate-putamen. The area of elevated brain lactate clearly exceeded the acidic core of the infarct and included the slightly alkaline border zone. The data demonstrate that after 7-hour middle cerebral artery occlusion, the reduction of the apparent diffusion coefficient in nuclear magnetic resonance diffusion images reflects precisely the region of histological injury, breakdown of energy metabolism, and tissue acidosis.

Previous therapeutic studies on the prevention of selective vulnerability of neurons in the hippocampus have suggested that the critical period for induction of delayed neuronal injury occurs early during recirculation. To determine the onset and duration of this period, an ultrashort-acting barbiturate (methohexital) was infused into the left carotid artery of 47 gerbils after various times of recirculation following 10 minutes of bilateral forebrain ischemia. Neuronal density in the left CA1 sector was determined 7 days later by counting the number of surviving neurons per millimeter of pyramidal cell layer. In 16 saline-treated gerbils, less than 10% of the CA1 neurons survived the 10 minutes of ischemia. Postischemic carotid infusion of methohexital improved neuronal survival, the degree of improvement depending on the timing and duration of the methohexital infusion. When carried out during the initial 40 minutes of recirculation, methohexital infusion for 10 minutes increased the number of surviving neurons to approximately 60% of that in five sham-operated control gerbils. This increase was significant for infusions carried out between the 10th and 20th minutes (n = 6, p less than 0.05) and between the 30th and 40th minutes (n = 6, p less than 0.05) of recirculation. Methohexital infusion for 20 minutes increased neuronal survival to 95% and 73% of that in the controls when carried out between the 0th and 20th minutes (n = 5, p less than 0.005) and between the 20th and 40th minutes (n = 5, p less than 0.005) of recirculation, respectively. Protection was nonsignificant for 10- or 20-minute methohexital infusions carried out after the 40th minute of recirculation. Our results demonstrate that the pathologic processes leading to delayed neuronal injury in the CA1 sector are induced during the initial 40 minutes of recirculation and that barbiturates are able to reverse these processes only if given during this period.

The four-vessel occlusion (4VO) model of Pulsinelli and Brierley (Stroke 1979;10:267-272) has been modified for use in halothane-nitrous oxide-anesthetized, physiologically controlled rats that were ventilating spontaneously. Selection criteria for the classification of severity of ischemia were established by correlating changes in the electroencephalogram and the general physiological status with measurements of regional blood flow and regional energy metabolism. In 13% of animals, 4VO did not cause flattening of the electroencephalogram, and such animals were classified as undergoing only "oligemia." In 65% of rats, the electroencephalogram flattened and blood pressure sharply increased with 4VO, whereas spontaneous respiration continued. This group exhibited almost complete ischemia in autoradiographic blood-flow studies, severe acidosis, and depletion of adenosine 5'-triphosphate and glucose in the forebrain and, hence, was classified as the "ischemia" group. The remaining 22% stopped breathing after vascular occlusion and were rejected for further study. Survival experiments of ischemic animals revealed the typical postischemic sequelae, with primary metabolic recovery after 8 hours of recirculation in all brain structures followed after 8-24 hours by severe biochemical deterioration and neuronal death in the striatum and hippocampus. Postischemic seizure activity was rare. The main advantages of the present modification in comparison with the original method are 1) the application of anesthesia without loss of primary selection criteria, 2) the possibility of invasive physiological monitoring, and 3) the absence of postischemic seizures, which eliminates the necessity for secondary selection criteria.

In lightly anesthetized cats, the left middle cerebral artery was occluded using a transorbital approach. Pial arterial pressure was measured with a feedback-controlled micropressure recording system in the territory of the occluded artery, and compared with changes of cortical blood flow, cortical steady potential and cortical EEG activity. After middle cerebral artery occlusion pial artery pressure fell from 56.2 +/- 1.6 to 7.8 +/- 0.4 mm Hg; during the following two hours it again slowly rose to about 15 mm Hg. Cortical heat conductance, as a measure of blood flow, decreased from 15.1 +/- 0.2 to 11.9 +/- 0.2 X 10(-4) cal X cm-1 X sec-1 X degrees C-1, and remained at this level throughout the observation period. Cortical steady potential shifted by 9.1 +/- 0.7 mV towards negativity, and EEG amplitude was reduced by about 50%. Pial arterial pressure correlated with blood flow, cortical steady potential and EEG amplitude, but not with EEG frequency. Autoregulation and CO2 reactivity of blood flow were disturbed after middle cerebral artery occlusion. Calculation of extra- and intracortical vascular resistances revealed that this disturbance was entirely due to intracortical vasoparalysis whereas the collateral vessels supplying the ischemic territory continued to react to both pressure and CO2 changes. Maintained vascular reactivity of collateral vessels, therefore, is a decisive factor for the efficiency of therapeutic blood flow improvement after acute middle cerebral artery occlusion.

Focal cerebral ischemia was produced in monkeys by injection of autologous clots into the left internal carotid artery. Regional protein synthesis was studied 2 hours after embolism by autoradiographic and biochemical evaluation of [3H]phenylalanine incorporation into brain proteins, and the results were correlated with electrophysiologic (electroencephalogram, evoked potentials) and light microscopic observations. Ischemic territories were clearly identified on autoradiograms as sharply demarcated areas with reduced radioactivity. The localization of regions with reduced protein biosynthesis correlated with the early postembolic suppression of evoked potentials but not with the (improved) functional state and the morphologic alterations at the end of the experiment. Suppression of amino acid incorporation, in consequence, is a long-lasting event that allows documentation of the initial embolic impact for at least 2 hours irrespective of the subsequent recovery process.

The effect of selective injury of hippocampal neurons on the consolidation of memory traces was studied in gerbils (meriones unguiculatus) after production of mild cerebral ischemia. The right carotid artery was permanently ligated, and animals without gross neurological deficits ("symptom-negative" gerbils) were selected. Eight days and eight weeks after vascular ligation, cell counts of hippocampal neurons were carried out and correlated with regional blood flow and the acquisition of operant behaviour. Eight days after carotid artery occlusion, learning behaviour was significantly impaired although the number of hippocampal neurons had not changed and blood flow had even increased above normal. After eight weeks, learning behaviour and blood flow were normal but now a significant loss of pyramidal neurons was present in the CA1 and CA2 sectors of the hippocampus. Our observations demonstrate that it is possible to detect subtle functional disturbances by appropriate behavioural investigation before manifestation of selective injury of the hippocampus. Recovery of integrative function, despite persistent cellular damage, provides further evidence for central nervous plasticity.

In 48 cases the left middle cerebral artery was occluded under light barbiturate anaesthesia using a transorbital approach. The animals were kept alive for 1, 2, and 4 hours after vascular occlusion. Regional cerebral blood flow was measured by the intracardiac microsphere injection technique before ischemia, 15 min after the onset of ischemia, and at the end of experiments. The density of regional ischemia was correlated with EEG changes and with the electrolyte, water and metabolite content of the same tissue samples in which blood flow was assessed. In the territory of the occluded middle cerebral artery, cortical blood flow decreased from 41.4 +/- 3.8 to 21.3 +/- 4.0 ml/100 g/min (means +/- SE), the actual flow rate depending on the individual efficacy of collateral blood supply. At flow rates below 10--15 ml/100 g/min, ischemia involved more than 50% of the middle cerebral artery territory, water and electrolyte homeostasis was severely disturbed and ischemic brain edema developed. Adenosine triphosphate decreased to about 60% of the control value at flow rates below 40 ml/100 g/min, but it remained at this level down to flow rates as low as 5 ml/100 g/min. EEG intensity -- but not EEG frequency -- decreased in parallel with blood flow, indicating that with increasing density of ischemia an increasing portion of the excitable neuropil was inhibited. The development of ischemic brain edema determined the further progression of ischemia. When blood flow decreased below the threshold for water and ion disturbance, ischemia was progressive (critical ischemia), but an amelioration of flow occurred in animals in which flow remained above this level (non-critical ischemia). In the contralateral hemisphere the EEG, blood flow, water and electrolyte content did not change significantly during the initial few hours of ischemia. Diaschisis, in consequence, was not a prominent feature during the early phase of infarct development.

In normothermic anesthetized cats cerebral blood flow was interrupted completely for one hour by arterial clamping and induced hypotension. The effect of ischemia on the ionic gradients of the cerebral cortex was assayed by determining total cortical electrolytes and by recording the activities of extracellular potassium ([K+i1e) and subarachnoid sodium ions ([Na+])s) with ion-sensitive electrodes. During ischemia [K+]e increased from 3.3+/-0.3 to 56+/-5.4 mEq per liter (means+/-SE) and [Na+]s decreased from 133+/-3.8 to 53+/-5.8 mEq per liter. When the brains were recirculated with blood after one hour's ischemia, [K+]e and [Na+]a gradually returned to normal within 45 minutes. The calculated intracellular uptake of sodium during ischemia amounted to 139 mEq per kilogram dry weight, whereas the intracellular release of potassium was only 64 mEq per kilogram. The increase in intracellular cation was accompanied by a movement of water from the extracellular into the intracellular compartment, causing a reversible shrinkage of the extracellular space from 18.9 to 8.5 vol %. The changes in ionic gradients were related to the development and resolution of ischemic brain swelling, and to the elctrophysiological events during and after ischemia.

In cats, the early development of ischemic brain edema was studied 1 to 4 hours after transorbital occlusion of the left middle cerebral artery (MCA). Two groups of animals were compared: those in which blood flow in the territory of the MCA decreased below the threshold of 10--15 ml/100 g/min (critical ischemia) and those in which it remained above this level (non-critical ischemia). In animals with critical ischemia, water content in the cortex of the MCA territory increased from 80.7 +/- 0.4 to 83.0 +/- 0.3 vol. % (means +/- SE) within 4 h. Edema was associated with an increase in tissue osmolality by 16--22 mosm/kg w.w., and a rise of sodium from 262 +/- 9 to 454 +/- 13 meq/kg d.w. and a decrease of potassium from 442 +/- 20 to 305 +/- 32 meq/kg d.w. The sodium/potassium ratio rose from 0.60 +/- 0.03 to 1.55 +/- 0.17. The water and electrolyte disturbances were accompanied by a major shift of extracellular fluid into the intracellular compartment, as evidenced by the increase in cortical impedance from 282 to 660 ohm/cm within 2 h. According to the Maxwell equation, this reflects a narrowing of the extracellular space from 19.8 to 11.4%. Brain volume was continuously monitored using an induction transducer; swelling began within a few minutes of vascular occlusion, and it continued throughout the 4 h observation period. During this time the blood-brain barrier remained intact as evidenced by the absence of Evans blue staining. Edema was associated with disturbances of the energy producing metabolism, but there was no strict correlation with either lactate or the concentration of high energy phosphates. In animals without critical ischemia, i.e. in which blood flow remained above 10--15 ml/100 g/min, edema was absent despite a distinct deterioration of the energy state of the brain. Edema was also absent in the border zone, in the territory of the posterior cerebral artery and in the contralateral hemisphere of animals with both critical and non-critical ischemia. It is concluded that the early ischemic brain edema following middle cerebral artery occlusion is of the cytotoxic type, that it develops at a flow rate below 10--15 ml/100 g/min, and that it is not strictly correlated with the energy state of the brain.

Stroke-prone spontaneously hypertensive rats with arterial blood pressure above 210 mmHg were taken for the present study after appearance of neurological symptoms. Regional cerebral blood flow, glucose metabolism, and protein synthesis rate were evaluated on the same brain section by means of triple-labelled autoradiographic techniques. Consecutive sections were used in the pictorial presentation of glucose, ATP, and serum protein extravasation. In addition, NADH-fluorescence was recorded. Two different patterns of hypertension-induced brain lesions could be distinguished: in two animals sharply demarcated cysts were visible in the cortical grey matter. In these animals no regional inhomogeneities of flow and metabolism were present remote from the infarct. In contrast, in three animals cysts were located in the white matter, leading to pronounced hemodynamic and metabolic disturbances throughout the brain. It is concluded that edema-induced brain swelling was the main cause for reduction in blood flow and metabolism.

In cats air embolism of the brain was produced by injecting 0.6 ml blood foam into the innominate artery proximal to the origin of both common carotid arteries. Air embolism caused transient ischemia of the brain, reaching a maximum within 1 min after injection. Resolution of the air embolism began a few minutes later and was completed within 15 min in the center and within 30 min in the border zone of the main supplying arteries. During this phase tissue perfusion was inhomogenous with reduced flow rates in some areas and reactive hyperemia up to 300% in others. This resulted in venous hyperoxia and a decrease of arteriovenous oxygen difference to as low as 2 ml/100 ml blood. Reactive hyperemia was accompanied by brain swelling and an increase in intracranial pressure from 3.6 +/- 1.2 to 12.3 +/- 2.0 mm Hg. The reason for hyperemia was a decrease of cortical pH which fell from 7.33 +/- 0.03 to 7.03 +/- 0.05, and which caused a dilation of pial arteries up to 260%. Immediately after embolism, the EEG flattened and oxygen consumption decreased. After normalization of flow, oxygen consumption returned to normal, but EEG only partially recovered. Air embolism had little effect on the water and electrolyte content of the brain, and produced very little damage to the blood-brain barrier.

A double tracer autoradiographic technique using 131I-iodo-antipyrine and 14C-deoxyglucose is presented for the simultaneous measurement of blood flow and cerebral glucose utilization in the same animal. 131I is a gamma emitting isotope with a half life of 8.06 days and can be detected with adequate resolution on standard autoradiographic films. Autoradiograms are made before and after decay of 131I; the time interval between the 2 exposures and the concentration of the 2 tracers is adjusted to avoid significant cross-contamination. In this way, 2 film exposures are obtained which can be processed quantitatively like single tracer autoradiograms. The validity of the method for the investigation of local coupling of flow and metabolism was tested under various physiological and pathophysiological conditions. Coupling was tight in barbiturate-anesthetized healthy animals, but not under halothane anesthesia where uncoupling occurred in various subcortical structures. Focal seizures induced by topical application of penicillin on the cortical surface led to a coupled increase of metabolism and flow in thalamic relay nuclei but not at the site of penicillin administration where increased glucose utilization was not accompanied by similar increase in blood flow. Both coupled and uncoupled increases in local glucose utilization were observed in spreading depression and in circumscribed areas of experimental brain tumors. The results obtained demonstrate that double tracer autoradiography allows allows the very precise local assessment of cerebral blood flow and glucose utilization, and, therefore, is particularly suited to the study of regional coupling processes under various experimental conditions.

The threshold of the relation between regional cerebral blood flow and regional cerebral protein synthesis was investigated in gerbils submitted to a 1-hour occlusion of the left common carotid artery. Blood flow was measured with [131I]iodoantipyrine and protein synthesis with [14C]leucine using double-tracer autoradiography and trichloroacetic acid wash-incubation for removal of nonincorporated tracer radioactivity. Specific activity of blood and brain leucine and [14C]leucine incorporation into brain proteins was also measured by conventional high-performance liquid chromatography to validate the autoradiographic approach. In control gerbils, gray matter blood flow ranged between 180 and 220 ml/100 g/min and fractional amino acid incorporation was approximately 80%. Unilateral carotid artery occlusion resulted in graded ischemia with blood flow between 10 and 100 ml/100 g/min. Regional cerebral protein synthesis gradually declined at blood flows of less than 100 ml/100 g/min and approached 0 at a blood flow of 40 ml/100 g/min. This threshold for complete suppression of protein synthesis is much higher than that for maintenance of tissue energy state and suggests that the size of an infarct after focal ischemia is determined by the suppression of protein synthesis rather than by the breakdown of energy metabolism.

Focal cerebral ischemia was produced in 16 cats by occluding the left middle cerebral artery (MCA) for 120 min. Cortical blood flow and pial artery pressure were determined prior to vascular occlusion and after 15, 60 and 120 min. At the end of the experiments (after 120 min MCA occlusion) heads were frozen in situ with liquid nitrogen. Cooled brains were cut into 0.5 cm thick slices. From these slices twenty-micron sections passing through the territory of the MCA were prepared in a cryostat and used in the pictorial presentation of glucose and ATP. NADH-fluorescence was recorded from the tissue slice, immersed in liquid nitrogen. In addition, tissue samples were taken from regions of interest and used for quantitative determination of biochemical substrates. In all but two animals permanent MCA occlusion led to disturbances in the energy-producing metabolism, as indicated by reduction in glucose and ATP, and increase in lactate. The regions exhibiting bright NADH-fluorescence were much smaller than those in which ATP was absent. In 6 animals NADH-fluorescence was not increased but even decreased in areas with disturbed energy-producing metabolism. A close correlation was obtained after comparing cortical blood flow measured 15 min after MCA occlusion with the area of ATP-depletion at the end of the experiments. However, the size of ATP-depletion did not correlate with flow measured 60 or 120 min after MCA occlusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult male gerbils were submitted to 5-minute cerebral ischemia by bilateral carotid artery occlusion. At the end of ischemia and at various recirculation times ranging from 15 to 120 minutes, brains were frozen in situ and the regional distribution of ATP, glucose, and tissue pH was studied on coronal cryostat sections by bioluminescent and fluoroscopic techniques. During ischemia ATP was completely depleted, glucose decreased to less than 10% of control, and regional tissue pH decreased from 7.04-7.09 to about 6.0. After the beginning of recirculation tissue pH and the regional content of metabolites exhibited a triphasic course. After 15 minutes pH returned to or even above normal, and ATP- and glucose-induced bioluminescence normalized. However, there was a secondary deterioration of both tissue acidosis and the metabolic state after 30 minutes. After longer recirculation times changes again improved and returned to normal within 2 hours. These changes were similar in all brain regions with the exception of the CA1 sector of the hippocampus, where the transient normalization of tissue pH was absent after 15 minutes of recirculation. This finding is in line with the previously observed microcirculatory insufficiency of this area and demonstrates that the CA1 sector of the hippocampus suffers more pronounced postischemic acidosis than other less vulnerable regions of the brain.

In adult normothermic cats cerebral blood flow was interrupted for 1 hour by clamping the innominate and subclavian arteries. Following ischemia the brains were recirculated with blood, and the coagulation system was investigated by measuring coagulation times and blood content of fibrinogen and platelets. Ischemia induced progressive consumption coagulopathy with an increase in coagulation times and a decrease of platelets and fibrinogen by more than 40%. Coagulopathy was accompanied by a respiratory distress syndrome with a significant increase in the alveolar-arterial carbon dioxide gradient from --3.3 to --13.5 mm Hg. A correlation was found between plasma fibrinogen concentration, cerebral blood flow and electrophysiological function, indicating that a relationship exists between the severity of postischemic coagulopathy and functional recovery following prolonged cerebral ischemia.

We measured total and regional cerebral blood flow (CBF, rCBF) and cerebral metabolic rate (CMR) of oxygen (O2), glucose (G), and lactate (L) levels for 4 h after 16 min global brain ischemia in rhesus monkeys with and without post-insult thiopental therapy. Eleven monkeys weighing 4-5 kg anesthetized with 1 percent halothane, 66 percent nitrous oxide and 33 percent oxygen, were subjected to 16 min global brain ischemia by a combination of trimethaphan hypotension (to a mean arterial pressure of 50 torr) and a high pressure (1500 torr) neck tourniquet. Post-ischemia, 7 monkeys were untreated (controls) and 4 received thiopental 90 mg/kg infused intravenously over 60 min, beginning at 5 min post-ischemia. Total CBF and rCBF were measured by continuous monitoring of cerebral venous (torcula) and parietal-occipital (external scintillation) 133Xe activity, respectively, after intra-innominate artery injection of 500 micronCi 133Xe in saline. In control monkeys, hyperemia in rCBF, but not in total CBF was observed at 6-7 min post-ischemia, whereas both total CBF and rCBF increased in thiopental treated monkeys. The hyperemia in thiopental treated monkeys coincided with an increase in CMRG without a proportional increase in CMRO2 or lactate levels. Indeed, CMRO2 was depressed in the first 30 min post-ischemia. At 30 min post-ischemia, CMRO2 rose to twofold greater than pre-ischemia in control monkeys, but only to pre-ischemic levels in thiopental treated monkeys. The data suggest that thiopental therapy improves distribution of brain blood flow and brain glucose uptake early post-ischemia and depresses CMRO2 later post-ischemia.