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Two patterns of transforming growth factor-beta1 (TGF-beta1) expression were identified in brains of normotensive rats following permanent occlusion of the middle cerebral artery (MCAO). First, a relative increase of TGF-beta1 mRNA by 37% was found at 12 h after MCAO in the ipsilateral cingulate cortex as compared to the homotopic contralateral region. The cingulate cortex is located distant from the ischemic territory. Treatment with the glutamate receptor antagonists MK-801 and NBQX did not reduce this expression (34% and 26% increase, respectively). Therefore, peri-infarct depolarization waves were probably not responsible for induction. Secondly, an increase of TGF-beta1 mRNA by 116% was found at 7 days after MCAO within infarcted tissue. This expression was not reduced by the glutamate receptor antagonists MK-801 (increase 140%) and NBQX (increase 137%), either. TGF-beta1 mRNA expression in the cingulate cortex at 12 h after MCAO is possibly mediated by neurons and astroglia and may support cell survival. Expression in the infarcted tissue at 7 days after MCAO is most likely related to the invasion of monocytes and may be involved in the downregulation of inflammatory events, in neoangiogenesis, and in formation of a glial scar around the infarct.

C57Black/6 and SV129 mice are widely used for the production of transgenic mutants in molecular stroke research but the ischemic susceptibility of these strains is influenced by differences in vascular anatomy and the responsiveness to excitotoxins and vasodilatory stimuli. To differentiate between these opposing effects on infarct size, the vascular territory of the two strains was correlated with the hemodynamic, metabolic, and morphological consequences of permanent middle cerebral artery (MCA) occlusion. The vascular anatomy was studied by latex infusion, brain infarction by vital staining, the size of the ischemic penumbra by imaging of ATP and protein synthesis, and blood flow by laser–Doppler flowmetry. In C57Black/6 mice the MCA-supplied vascular territory and the size of brain infarcts were significantly larger than in SV129 mice but the size of the penumbra and the residual blood flow in the center of the MCA-supplying territory were similar in both strains. These findings suggest that differences in infarct size in C57Black/6 and SV129 mice are determined mainly by the vascular anatomy and not by differences in collateral vascular responsiveness or excitotoxicity.

The effects of a traumatic neocortical lesion on c-fos, junB, c-jun, MKP-1 and hsp72 expression were examined by in situ hybridization and immunocytochemistry 1-6 h following transcranial cold injury. The direct current potential was recorded in the injury-remote cortex to evaluate the role of transient direct current shifts, i.e. spreading depressions, in gene expression. In 14 out of 21 injured rats, spreading depression-like depolarizations of the direct current potential were noticed, which were accompanied by a transient decrease in electroencephalographic activity and increase in laser Doppler flow. In seven injured animals, no spontaneous spreading depressions were seen. In animals without spreading depressions, only a short-lasting response of c-fos, junB, c-jun and MKP-1 messenger RNAs as well as c-Fos protein was bilaterally found in the piriform cortex, and--with ipsilateral dominance--the dentate gyrus and hippocampal CA3/4 fields at 1 h after lesioning. In injured animals with spreading depressions however, a strong elevation was seen in layers II-IV and VI of the injury-remote ipsilateral cerebral cortex, which persisted over as long as 6 h. Messenger RNA levels for c-fos, junB and MKP-1 were closely related to the time interval between the last depolarization and the end of experiment. Levels were highest shortly after transient direct current shifts, and decreased thereafter mono-exponentially with half-lives of 48, 75 and 58 min for c-fos, junB and MKP-1 messenger RNAs, respectively. In 6 h animals with spreading depressions, hsp72 messenger RNA was slightly elevated in layer II of the injury-remote cortex, but heat shock protein 72 was not increased. The present results demonstrate that spreading depression is the most prominent factor influencing the trauma-related gene response in the lesion-remote cortical tissue.

Administration of inhibitors of neuronal nitric oxide synthase or deletion of the encoding gene in rodents provided evidence that neuronal nitric oxide synthase activity may contribute to neuronal cell death following global and focal cerebral ischemia. In the present study, we investigated by in situ hybridization the expression of an endogenous inhibitor of neuronal nitric oxide synthase activity, designated protein inhibitor of neuronal nitric oxide synthase and homologous to cytoplasmic dynein light chain, in the post-ischemic rat brain. Following global ischemia induced by cardiac arrest, messenger RNA expression of protein inhibitor of neuronal nitric oxide synthase was rapidly induced in pyramidal neurons of the hippocampal CA3 region and granule cell of the dentate gyrus which are resistant to ischemic damage. In vulnerable CA1 pyramidal neurons however, protein inhibitor of neuronal nitric oxide synthase expression remained at basal level after global ischemia and was associated with an increase in nicotinamide adenine dinucleotide phosphate-diaphorase activity and subsequent DNA fragmentation indicating ischemia-mediated neuronal cell death. Following focal cerebral ischemia induced by permanent occlusion of the middle cerebral artery, transcripts of protein inhibitor of neuronal nitric oxide synthase progressively accumulated in cortical neurons bordering the infarct area. After transient middle cerebral artery occlusion however, messenger RNA levels of protein inhibitor of neuronal nitric oxide synthase increased in the reperfused neocortex. Our findings indicate that cerebral ischemia leads to an increase in neuronal expression of protein inhibitor of neuronal nitric oxide synthase in brain regions where sustained or “uncoupled” nitric oxide synthase activity may be detrimental to neurons. Lack of post-ischemic induction of protein inhibitor of neuronal nitric oxide synthase in CA1 pyramidal neurons may result in high nitric oxide synthase activity after global ischemia and could contribute to delayed neuronal cell death.

The effect of the glutamate antagonist MK-801 on the ischemic threshold of energy metabolism and protein synthesis (CPS) was studied in rats submitted to 3 h occlusion of the left middle cerebral artery (MCA). Local blood flow and CPS were measured by double tracer autoradiography, and local ATP content by bioluminescence imaging. In untreated animals breakdown of energy metabolism occurred at flow values below 15 +/- 1 and CPS inhibition below 51 +/- 15 ml/100 g/min (means +/- S.D.). MK-801 treatment (3 mg/kg immediately after MCA occlusion) did not change the ischemic flow threshold of energy failure (16 +/- 3 ml/100 g/min) but lead to a highly significant decline of the perfusion threshold for the inhibition of CPS to 19 +/- 4 ml/100 g/min (P0.01). Our data demonstrate that MK-801 dramatically reduces the threshold for the suppression of protein synthesis which could explain previously reported therapeutical effects on the reduction of brain infarct size.

The effect of manganese(III)tetraphenylporphine sulfonate (MnTPPS) on the relaxation enhancement of NMR images (MRI) was studied in experimental brain tumors in rats. Brains were inoculated with the glioma cell line F98 12 to 19 days before the NMR experiment, and the effect of MnTPPS (0.25 mmol/kg body weight) was investigated 2 and 4 days after intraperitoneal injection. After MnTPPS addition tumors could be clearly distinguished by the brightness from the surrounding brain whereas they were barely visible without contrast enhancement. At SE time of 25 msec and TR time of 3500 msec the ratio of magnetization values of tumor versus normal grey matter increased from 0.98 ± 0.08 to 1.24 ± 0.09 (means ± SD). When TR was shortened to 1100 msec contrast enhancement further increased to 1.77 ± 0.25. These results demonstrate for the first time that MnTPPS is an efficient agent for contrast enhancement of brain tumors.

Transient forebrain ischemia of 30 min duration was produced in anaesthetized rats by four-vessel occlusion. After survival periods of 3 h to three days brains were perfusion-fixed and sections through the mid-dorsal hippocampus were processed for conventional staining and immunohistochemical analysis. Neuronal damage in the hilus was manifested 3-8 h after ischemia; neurons in the CA1 and CA2 sector suffered delayed neuronal death after 48-72 h whereas the dentate gyrus and the CA3 sector were normal. Vasogenic edema formation was visualized using antibodies against rat serum-proteins, serum albumin and immunoglobulins. By 3 h after ischemia, only faint and diffuse serum-staining was detected. At 8 h survival, weak astrocytic-staining was present. After 24-72 h CA1-CA2 exhibited massive serum extravasation. The molecular layer of the dentate gyrus showed edema formation in the absence of granule cell damage. The glial reaction was studied using antibodies against glial fibrillary acidic protein, vimentin and S-100 protein. Glial fibrillary acidic protein and S-100 protein-staining increased in areas with either edema or neuronal damage. In contrast, changes in vimentin were only detected in areas with neuronal necrosis. The observations demonstrate that following 30 min of ischemia neuronal damage is accompanied by changes in blood-brain barrier function and reactive glial alterations. The dissociation between neuronal necrosis and astroglial hypertrophy and hyperplasia reflects differences in cellular responsiveness which constitute inherent features of postischemic hippocampal injury.

The blood-brain transport and regional distribution of a tritium-labeled, brominated benzodiazepine (BFB) was determined for the rat brain in vivo. The unidirectional transport constant from blood to brain was measured by a graphical, integral method and was found to be 0.83 ml/g/min, a value which indicates that transport is essentially flow-dependent. The apparent volume of distribution increased linearly during the measurement period, suggesting that back transport from brain to blood was zero and that BFB was trapped in the tissue, possibly by specific receptors or acceptors. Under the conditions of these experiments, autoradiography of brain tissue sections indicated a regional distribution of [ 3H]BFB similar to that expected for regional cerebral blood flow. These results indacate that BFB is a useful blood flow tracer in brain and suggest that BFB radiobrominated with bromine-75 may, under appropriate conditions, be a suitable tracer for in vivo regional blood flow management or benzodiazepine receptor mapping by positron-emission tomography.

A technique was developed for the quantitative regional assessment of brain pH by a modification of the umbelliferone method. Twenty micron thick sections were brought into contact with umbelliferone-soaked paper strips and the fluorescence (450 nm) following excitation at 370 nm and 340 nm was recorded photographically. The 340 nm excitation image was subtracted from 370 nm picture, using a computerized image-processing system. Regional pH values were measured in rats and cats under normal and ischemic conditions.

Brain tissue osmolality was measured in cats 2 h after permanent middle cerebral artery occlusion. Blood osmolality was deliberately varied by various therapeutic procedures from 306 to 375 mosm. Osmolality of the blood, the spinal fluid, and both ischemic and nonischemic brain tissue were well equilibrated but there were consistent differences between the various compartments: ischemic tissue osmolality (342 ± 22 mosm, mean ± SD) was about 15 mosm higher than that of nonischemic tissue (327 ± 23 mosm), and cerebral spinal fluid osmolality (333 ± 18 mosm) was 6 mosm higher than nonischemic but 9 mosm lower than ischemic brain tissue. A direct correlation existed between tissue osmolality and brain water, and inverse correlations with electroencephalogram (EEG) intensity, the size of the extracellular space, and the ratio of Na:K concent of ischemic brain tissue. Ischemic brain water increase could be prevented by either increasing blood flow to more than 25 ml 100 g−1 min−1 or by increasing blood osmolality above 335 mosm/liter.

Transient cerebral ischaemia of 20 min to 2 h was produced in normothermic cats by clamping the innominate and subclavian arteries and simultaneous lowering of the systemic blood pressure, or by interrupting the blood supply to animals with extracorporeal circulation. After ischaemia high systemic blood pressure was maintained to assure the adequate blood recirculation of the brain. The effect of ischaemia on the sensorimotor cortex was studied by recording the spontaneous EEG and the pyramidal response (PR) to electrical stimulation of the cortex. When ischaemia was produced by clamping the innominate and subclavian arteries the EEG was suppressed after 12.8 ± 3.8 sec, the I wave of the PR after 181 ± 51 sec, and the D wave after 261 ± 50 sec. In the animals with extracorporeal circulation the suppression time of the EEG was 16.9 ± 5.7 sec, of the I wave 178 ± 37 sec and of the D wave 271 ± 50 sec. Under optimal conditions the D wave reappeared as early as 7 min after ischaemia of 1 h, the I wave after 25 min and the EEG after 45 min. Even after ischaemia of 1.5 h the I and D waves reappeared, and the D wave transitorily after ischaemia of 2 h. The results confirm that basic neuronal functions may reappear after complete ischaemia of more than 1 h.

A method is described by means of which the midline of the cat brain can be split without severing the corpus callosum (“corpus callosum cat”). This procedure provides a preparation for the study of interhemispheric volleys, in which the only anatomical connection between the two hemispheres remains the corpus callosum.

Regional changes of cerebral blood flow and biochemical substrates were assessed in the gerbil brain following different grades of cerebral ischemia. Ischemia was produced by occlusion of the right common carotid and left external carotid arteries. Gerbils were classified according to the severity of neurological symptoms as animals without, with mild and with severe neurological deficits. Brains were frozen in situ, sliced in 20-μm sections and processed for pictorial presentation of glucose and ATP, using bioluminescence techniques. Cerebral blood flow was determined in adjacent brain sections, using [ 14 C]iodoantipyrine autoradiography. NADH fluorescence was recorded by illuminating the surface of the tissue block with ultraviolet light. Most animals without visible neurological symptoms exhibited reduced blood flow in circumscribed regions of cortex and basal ganglia of the right hemisphere without concomitant changes of biochemical substrates. In animals with mild neurological symptoms, blood flow in the right hemisphere was reduced, glucose and ATP decreased, and NADH fluorescence inhomogeneously enhanced. In animals with severe neurological symptoms blood flow was almost arrested in the right hemisphere and was distinctly reduced in the medial parts of the left hemisphere. The ischemic tissue was depleted from glucose and ATP, and exhibited bright NADH fluorescence. The severity of neurological symptoms, in consequence, correlated closely with both the degree and the size of biochemical lesions observed in the ischemic territory.