The cytosolic antioxidant, copper/zinc superoxide dismutase, attenuates blood-brain barrier disruption and oxidative cellular injury after photothrombotic cortical ischemia in mice.

Oxidative stress has been associated with the development of blood-brain barrier disruption and cellular injury after ischemia. The cytosolic antioxidant, copper/zinc superoxide dismutase, has been shown to protect against blood-brain barrier disruption and infarction after cerebral ischemia-reperfusion. However, it is not clear whether copper/zinc superoxide dismutase can protect against evolving ischemic lesions after thromboembolic cortical ischemia. In this study, the photothrombotic ischemia model, which is physiologically similar to thromboembolic stroke, was used to develop cortical ischemia. Blood-brain barrier disruption and oxidative cellular damage were investigated in transgenic mice that overexpress copper/zinc superoxide dismutase and in littermate wild-type mice after photothrombotic ischemia, which was induced by both injection of erythrosin B (30 mg/kg) and irradiation using a helium neon laser for 3 min. Free radical production, particularly superoxide, was increased in the lesioned cortex as early as 4 h after ischemia using hydroethidine in situ detection. The transgenic mice showed a prominent decrease in oxidative stress compared with the wild-type mice. Blood-brain barrier disruption, evidenced by quantitation of Evans Blue leakage, occurred 1 h after ischemia and gradually increased up to 24 h. Compared with the wild-type mice, the transgenic mice showed less blood-brain barrier disruption, a decrease in oxidative DNA damage using 8-hydroxyguanosine immunohistochemistry, a subsequent decrease in DNA fragmentation using the in situ nick-end labeling technique, and decreased infarct volume after ischemia. From these results we suggest that superoxide anion radical is an important factor in blood-brain barrier disruption and oxidative cellular injury, and that copper/zinc superoxide dismutase could protect against the evolving infarction after thromboembolic cortical ischemia.

Progressive and reproducible focal cortical ischemia with or without late spontaneous reperfusion generated by a ring-shaped, laser-driven photothrombotic lesion in rats.

Clinical stroke is mostly of thromboembolic origin, in which the magnitude of brain damage resulting from arterial occlusions depends on the degree and duration of the concomitant ischemia. To facilitate more controllable and reproducible study of stroke-related pathophysiological mechanisms, a photothrombotic ring stroke model was initially developed in adult rats. The ring interior zone comprises an anatomically well confined cortical region-at-risk which is gradually encroached by progressive hypoperfusion, thus mimicking the situation (albeit in inverse fashion) of an ischemic penumbra or stroke-in-evolution. Modification of this model using a thinner ring irradiation beam resulted in late spontaneous reperfusion in the cortical region-at-risk and a remarkable morphological tissue recovery in this ostensibly critically injured region. On the other hand, doubling the thin irradiating beam intensity facilitates a complementary situation in which lack of reperfusion in the region-at-risk after stroke induction leads to tissue pannecrosis. The dual photothrombotic ring stroke model, effectuated either with or without reperfusion and thereby tissue recovery or pannecrosis, may be well suited for the study of events related to postischemic survival or cell death in the penumbra region. To popularize the photothrombotic ring stroke model, we present a detailed protocol of how this model is induced in either version as well as protocols for transcardial carbon black perfusion and laser-Doppler flowmetry experiments.

Thromboembolic events lead to cortical spreading depression and expression of c-fos, brain-derived neurotrophic factor, glial fibrillary acidic protein, and heat shock protein 70 mRNA in rats.

The hypotheses that cerebral embolic events lead to repetitive episodes of cortical spreading depression (CSD) and that these propagating waves trigger the expression of c-fos, brain-derived neurotrophic factor (BDNF), glial fibrillary acidic protein (GFAP), and heat shock protein 70 (HSP70) mRNA were tested. Wistar rats underwent photochemically induced right common carotid artery thrombosis (CCAT) (n = 18) or sham (n = 8) procedures. In a subgroup of rats (n = 5), laser-Doppler flowmetry probes were placed overlying the right parietal cortex to record CSD-like changes in cortical blood flow during the initial 2-hour postinjury period. Rats were killed by decapitation at 2 or 24 hours after CCAT, and brains were processed for in situ localization of the gene expression. Two to five intermittent transient hyperemic episodes lasting 1 to 2 minutes were recorded ipsilaterally after CCAT. At 2 hours after CCAT, the widespread expression of c-fos and BDNF mRNAs was observed throughout the ipsilateral cerebral cortex. Pretreatment with the N-methyl-D-aspartate receptor blocker MK-801 (2 mg/kg) 1 hour before CCAT reduced the expression of BDNF mRNA expression at 2 hours. At 24 hours after CCAT, increased expression of GFAP mRNA was present in cortical and subcortical regions. In contrast, multifocal regions of HSP70 expression scattered throughout the thrombosed hemisphere were apparent at both 2 and 24 hours after injury. These data indicate that thromboembolic events lead to episodes of CSD and time-dependent alterations in gene expression. The ability of embolic processes to induce widespread molecular responses in neurons and glia may be important in the pathogenesis of transient ischemic attacks and may influence the susceptibility of the postembolic brain to subsequent insults including stroke.

Delayed hypovolemic hypotension exacerbates the hemodynamic and histopathologic consequences of thromboembolic stroke in rats.

Abnormalities in cerebrovascular reactivity or hemodynamic reserve are risk factors for stroke. The authors determined whether hemodynamic reserve is reduced in an experimental model of thromboembolic stroke. Nonocclusive common carotid artery thrombosis (CCAT) was produced in rats by a rose bengal-mediated photochemical insult, and moderate hypotension (60 mm Hg/30 min) was induced 1 hour later by hemorrhage. Alterations in local cerebral blood flow (ICBF) were assessed immediately after the hypotensive period by 14C-iodoantipyrine autoradiography, and histopathologic outcome was determined 3 days after CCAT. Compared to normotensive CCAT rats (n = 5), induced hypotension after CCAT (n = 7) led to enlarged regions of severe ischemia (i.e., mean ICBF < 0.24 mL/g/min) in the ipsilateral hemisphere. For example, induced hypotension increased the volume of severely ischemic sites from 16 +/- 4 mm3 (mean +/- SD) to 126 +/- 99 mm3 (P < 0.05). Histopathologic data also showed a larger volume of ischemic damage with secondary hypotension (n = 7) compared to normotension (22 +/- 15 mm3 versus 5 +/- 5 mm3, P < .05). Both hypotension-induced decreases in ICBF and ischemic pathology were commonly detected within cortical anterior and posterior borderzone areas and within the ipsilateral striatum and hippocampus. In contrast to CCAT, mechanical ligation of the common carotid artery plus hypotension (n = 8) did not produce significant histopathologic damage. Nonocclusive CCAT with secondary hypotension therefore predisposes the post-thrombotic brain to hemodynamic stress and structural damage.

Ritanserin, a 5-HT2 receptor antagonist, increases subcortical blood flow following photothrombotic middle cerebral artery occlusion in rats.

It has been proposed that the reversal of serotonin-mediated vasoconstriction accounts for the neuroprotective effect of serotonin (5-HT2) receptor blockade in focal cerebral ischemia. We investigated the effect of pretreatment with ritanserin, a 5-HT2 receptor antagonist, on cerebral blood flow in a model of photothrombotic middle cerebral artery occlusion in rats. Local cerebral blood flow was measured by iodoantipyrine autoradiography 30 minutes after induction of ischemia. Using a novel image-alignment algorithm, 3-dimensional reconstructions of averaged cerebral blood flow were calculated. The difference-image of local cerebral blood flow between ritanserin and vehicle-treated animals revealed a subcortical zone underlying the ischemic cortex where cerebral blood flow was markedly enhanced indicating a beneficial hemodynamic effect of ritanserin. Three-dimensional image analysis provides a powerful tool to detect inter-group differences of cerebral blood flow which are underestimated by conventional types of data analysis.

Usual and unusual methods for detection of lipid peroxides as indicators of tissue injury in cerebral ischemia: what is appropriate and useful?

1. Free radical-dependent lipid peroxidation processes have long been thought to contribute to brain damage following stroke or cerebral ischemia/reperfusion. 2. The preponderance of evidence for this belief has been derived indirectly, through diminution of tissue injury indices (e.g., brain infarct volume) facilitated by application of free radical scavenger substances. 3. Direct, unequivocal evidence for lipid peroxidation in terms of classical assays (detection of conjugated diene absorbance or thiobarbituric acid-reactive substances) is considerably less common, and its validity can be questioned. 4. Correlations of treatment-induced diminishment of brain injury indices with reductions in lipid peroxidation level are rarer still. 5. Reasons underlying the disparity between the belief that lipid peroxidation contributes to ischemic brain injury and direct evidence for this contribution (at least acutely) are proposed, along with evidence that new methods are being developed which should provide the basis for obtaining a definitive answer.

Macroscopic indices of lipid peroxidation in cerebral ischemia/reperfusion: validity and sensitivity enhancement in terms of conjugated diene detection.

Recent evidence for the occurrence of in vivo lipid peroxidation in the context of cerebral ischemia/reperfusion, as detected by classical tests such thiobarbituric acid reactivity and conjugated diene absorbance, is critically reviewed. Despite the widespread perception that lipid peroxidation is well-established and to be expected under such circumstances, in general these detection methods have not been applied with rigor sufficient to prove the quantitative existence of lipid peroxides unequivocally. The development of sensitive methods which can be utilized in small tissue samples at early times after brain injury is needed. In particular, the conditions necessary for the establishment of a more rigorous and sensitive method of conjugated diene detection in terms of difference spectral analysis are detailed and illustrated. In addition, a new autofluorescence in the far-ultraviolet region is shown to be associated with oxygenated conjugated diene-containing compounds.

L-arginine does not improve cortical perfusion or histopathological outcome in spontaneously hypertensive rats subjected to distal middle cerebral artery photothrombotic occlusion.

The potential of nitric oxide (NO) to influence positively or negatively the outcome of mechanically induced focal cerebral ischemia is still controversial. Recent evidence suggests that NO of vascular origin, whether synthesized from exogenously administered L-arginine (L-Arg) or from NO donor compounds, is beneficial but that of neuronal origin is not. However, the therapeutic potential of NO to ameliorate stroke induced by arterial thrombosis has not been reported. We assessed the therapeutic effect of L-Arg administration in spontaneously hypertensive rats (SHR) subjected to permanent photothrombotic occlusion of the distal middle cerebral artery (dMCA). The ipsilateral carotid artery was left unligated to enhance L-Arg delivery into the putative penumbral region. Local CBF (LCBF) was assessed at 30 min by the [14C]iodoantipyrine technique (n = 9), while histological infarct volumes and index of peripheral ischemic cell change were determined at 3 days (n = 7). Rats (n = 9) given 300 mg/kg L-Arg at 18 and 3 h before photothrombotic dMCA occlusion and at 5 min afterward displayed no significant differences in LCBF compared with animals (n = 8) injected with water (the carrier vehicle) and similarly irradiated. Infarct volumes were also similar, being 37.0 +/- 9.7 mm3 (SD) in the vehicle-treated and 49.1 +/- 17.2 mm3 (SD) in the L-Arg-treated groups (both n = 7), as were assessments of ischemic neuronal density in the penumbra. In contrast, L-Arg administered intravenously in a dose of 300 mg/kg to nonischemic SHR (n = 5) increased cortical CBF by approximately 75% during a 70-min observation period. We conclude that thrombotic processes superimposed upon cerebral ischemia may facilitate tissue reactions that offset the potentially beneficial effect of L-Arg, and this caveat must be considered when proposing L-Arg for clinical treatment of focal thrombotic stroke.

Induction of spreading depression in the ischemic hemisphere following experimental middle cerebral artery occlusion: effect on infarct morphology.

This study was undertaken to test whether transient depolarizations occurring in periinfarct regions are important in contributing to infarct spread and maturation. Following middle cerebral artery (MCA) occlusion we stimulated the ischemic penumbra with recurrent waves of spreading depression (SD) and correlated the histopathological changes with the electrophysiological recordings. Halothane-anesthetized, artificially ventilated Sprague-Dawley rats underwent repetitive stimulation of SD in intact brain (Group 1; n = 8) or photothrombotic MCA occlusion coupled with ipsilateral common carotid artery occlusion (Groups 2 and 3, n = 9 each). The electroencephalogram and direct current (DC) potential were recorded for 3 h in the parietal cortex, which represented the periinffarct border zone in ischemic rats. In Group 2, only spontaneously occurring negative DC shifts occurred; in Group 3, the (nonischemic) frontal pole of the ischemic hemisphere was electrically stimulated to increase the frequency of periinfarct DC shifts. Animals underwent perfusion-fixation 24 h later, and volumes of complete infarction and scattered neuronal injury ("incomplete infarction") were assessed on stained coronal sections by quantitative planimetry. Electrical induction of SD in Group 1 did not cause morphological injury. During the initial 3 h following MCA occlusion, the number of spontaneous periinfarct depolarization in Group 2 (7.0 +/- 1.5 DC shifts) was doubled in Group 3 by frontal current application (13.4 +/- 2.7 DC shifts; p < 0.001). The duration as well as the integrated negative amplitude of DC shifts over time were significantly greater in Group 3 than in Group 2 rats (duration, 5.7 +/- 3.8 vs. 4.1 +/- 2.5 min; p < 0.05). Histopathological examination disclosed well-defined areas of pannecrosis surrounded by a cortical rim exhibiting selectively damaged acidophilic neurons and astrocytic swelling in otherwise normal-appearing brain. Induction of SD in the ischemic hemisphere led to a significant increase in the volume of incomplete infarction (19.0 +/- 6.1 mm3 in Group 3 vs. 10.3 +/- 5.1 mm3 in Group 2; p < 0.01) and of total ischemic injury (100.7 +/- 41.0 mm3 in Group 3 vs. 66.5 +/- 24.7 mm3 in Group 2; p < 0.05). The integrated magnitude of DC negativity per experiment correlated significantly with the volume of total ischemic injury (r = 0.780, p < 0.0001). Thus, induction of SD in the ischemic hemisphere accentuated the development of scattered neuronal injury and increased the volume of total ischemic injury. This observation may be explained by the fact that with limited perfusion reserve, periinfarct depolarization are associated with episodic energy failure in the acute ischemic penumbra.

Neurobehavioral consequences of induced spreading depression following photothrombotic middle cerebral artery occlusion.

In a model of experimental focal cerebral ischemia, we have recently reported a strong correlation between the magnitude of ischemic depolarizations in the peri-infarct borderzone and the extent of histological injury. In the present study, we assessed the neurobehavioral consequences of spontaneously occurring and induced ischemic depolarizations in rats following middle cerebral artery (MCA) occlusion, as well as the effects of induced spreading depression (SD) in intact animals. Halothane-anesthetized, artificially ventilated Sprague-Dawley rats underwent photothrombotic MCA occlusion coupled with ipsilateral common carotid artery (CCA) occlusion. The electroencephalogram and direct current (DC) potential were recorded in the parietal infarct borderzone-corresponding to the cortical forelimb area-for 3 h following MCA occlusion. Group 1 rats (n = 9) received MCA/CCA occlusion, and the spontaneously occurring negative DC shifts were recorded in the ischemic borderzone. In Group 2 animals (n = 9), the (non-ischemic) frontal pole of the ipsilateral hemisphere was electrically stimulated in order to double the frequency of peri-infarct DC shifts occurring over the initial 3 h postocclusion. Group 3 consisted of intact rats (n = 3) in which SD was repeatedly evoked in the frontal pole. Four animals served as sham-operated controls. A battery of sensorimotor behavioral tests, consisting of beam balance, postural reflex and elicited forelimb placing, was applied in a blinded fashion. Sham controls and animals of Groups 1 and 2 were tested 24 h after surgery, and Group 3 rats were tested 2, 6 and 24 h after generation of SDs. A cumulative neurobehavioral index, ranging from 0 to 144, was calculated by adding the individual test results. Brains were perfusion-fixed 24 h following surgery for calculation of volumes of infarction and scattered neuronal injury. Functional outcome at 24 h was significantly worse in Group 2 animals (spontaneous plus induced ischemic depolarizations) (neurobehavior index 43 +/- 19, mean +/- S.D.) compared to Group 1 rats, in which only spontaneous depolarizations occurred (neurobehavior index 24 +/- 19, P < 0.05). The cumulative neurobehavioral index of Group 1 and 2 animals correlated positively with the volume of total ischemic injury (r = 0.765, P < 0.001) and with the frequency of ischemic depolarizations (r = 0.474, P < 0.05). Correlations between severe forelimb placing deficits and severe degrees of histological injury (necrosis or ischemic cell change) in the corresponding primary sensorimotor cortical region FR1 were significant in these rats. Group 3 rats showed severe neurobehavioral deficits at 2 and 6 h following SD stimulation (index 57 +/- 1 and 39 +/- 1, respectively) but returned to normal at 24 h (4 +/- 0). The findings indicate that cortical spreading depression is accompanied by transient neurobehavioral deterioration and that SD in the ischemic hemisphere of animals subjected to MCA occlusion worsened functional outcome 24 h after surgery.

Nonocclusive common carotid artery thrombosis in the rat results in reversible sensorimotor and cognitive behavioral deficits.

BACKGROUND AND PURPOSE: Microemboli released during transient ischemic attack, stroke, and cardiac surgery are thought to cause a variety of functional deficits in humans. The purpose of this study was to characterize the type and extent of neurobehavioral deficits present after photochemically induced common carotid artery thrombosis (CCAT), a thromboembolic model of stroke in the rat that results in a platelet emboli shower. METHODS: Thirty-two male Wistar rats were assigned to four groups. Groups 1 (n = 8) and 3 (n = 8) were long-term (6-week survival) and short-term (2-week survival) experimental groups subjected to right CCAT with the use of the photochemical technique. Groups 2 (n = 8) and 4 (n = 8) served as sham-operated controls for each experimental group. A battery of behavioral tests was applied daily beginning 24 hours after thrombosis; this consisted of elicited forelimb placing, postural reflex, beam balance, beam walking, and open field activity. Cognitive testing with a water maze task was performed on post-CCAT days 30 to 33 for groups 1 and 2 and on post-CCAT day 2 for groups 3 and 4. Ten-micrometer coronal brain sections were stained with hematoxylin and eosin, and infarct location and frequency were determined. RESULTS: Significant sensorimotor deficits were observed, which recovered within 2 weeks after CCAT. The data that follow are derived by combining the two experimental groups and comparing these with the two sham groups. The following tests showed significant effects after CCAT: contralateral elicited forelimb placing, ipsilateral elicited forelimb placing, beam balance, and beam walking score. Cognitive dysfunction was seen acutely (group 3 animals) at 2 days after CCAT; Morris water maze length and latency to target were significantly greater in the experimental group. No deficits were seen in postural reflex, open field activity, or delayed cognitive testing. Histopathological assessment revealed small infarcts in 11 of 16 thrombosed rats. However, a strong relationship between neurobehavioral deficits and infarct location was not consistently demonstrated. CONCLUSIONS: CCAT produces consistent sensorimotor and cognitive behavioral deficits that recover within 2 weeks of injury. Behavioral outcome was not necessarily associated with overt histopathological damage, suggesting that reversible injury mechanisms, both vascular and neuronal, may be partly responsible for the temporary loss of function. These data strengthen the role of CCAT as a clinically relevant model of thromboembolic stroke.

Post-ischemic administration of HU-211, a novel non-competitive NMDA antagonist, protects against blood-brain barrier disruption in photochemical cortical infarction in rats: a quantitative study.

We examined the effect of HU-211, a synthetic non-psychotropic cannabinoid with non-competitive N-methyl-D-aspartate (NMDA) antagonist properties, on blood-brain barrier (BBB) integrity after photochemically induced cortical infarction. Evans blue dye was used as a BBB permeability indicator after unilateral thrombotic cortical infarction was produced photochemically by 560 nm light irradiation of the cortex in male Wistar rats receiving rose bengal intravenously. HU-211 was injected in a dose of 4 mg/kg i.v. 30 min after stroke. Fluorometric measurement of Evans blue was performed 24 h later in six brain regions. Treatment with HU-211 significantly decreased extravasation of dye into the area of infarct (406 +/- 19 vs. 539 +/- 33 micrograms/g, mean +/- S.E.M.) as well as other sites of the affected hemisphere (866 +/- 68 vs. 1096 +/- 68 micrograms/g) compared to the vehicle group. These data indicate that HU-211 is an effective drug in protecting against the effects of focal ischemia-induced BBB disruption in the rat and suggest that the drug may be an effective treatment against the ischemic cell death and BBB disruption that can occur clinically following a stroke or cardiac arrest.

Local cerebral glucose utilization and cytoskeletal proteolysis as indices of evolving focal ischemic injury in core and penumbra.

To ascertain the tempo of progression to irreversible injury in focal ischemia, we subjected halothane-anesthetized Sprague-Dawley rats to photochemically induced distal middle cerebral artery occlusion (dMCAO) combined with permanent ipsilateral and 1 h contralateral common carotid artery occlusions. Head temperature was maintained at 36 degrees C. At times centered at either 1.5 or 3 h post-dMCAO, the rate of local glucose metabolism (lCMRgl) was measured by 2-deoxyglucose autoradiography, and cytoskeletal proteolysis was assessed regionally by an immunoblotting procedure to detect spectrin breakdown products. At 1.5 h (n = 5), the cortical ischemic core was already severely hypometabolic (lCMRgl 15.5 +/- 10.8 mumol 100 g-1 min-1, mean +/- SD), whereas the cortical penumbral zone was hypermetabolic (69.0 +/- 9.7). (The lumped constant was verified to be unchanged by methylglucose studies). Neutral red pH studies at this time point showed that both the core and penumbral zones were equally acidotic. By 3 h post-dMCAO (n = 6), lCMRgl in the penumbral zone had fallen to low levels (15.4 +/- 2.2 mumol 100 g-1 min-1) equal to those of the ischemic core (16.7 +/- 4.5). Correspondingly, spectrin breakdown in the ischemic core was advanced at both 2 and 3.5 h post-dMCAO (36 +/- 18% and 33 +/- 18% of total spectrin, respectively), whereas in the penumbral zone spectrin breakdown was less extensive and more highly variable at both times (22 +/- 23% and 29 +/- 16%). We conclude that irreversible deterioration of the ischemic core, as evidenced by the onset of local cytoskeletal proteolysis, begins within 2 h of middle cerebral artery occlusion. In the ischemic penumbra, the transition from glucose hyper- to hypometabolism occurs by 3.5 h and is associated with a milder and more variable degree of spectrin breakdown.

A photothrombotic 'ring' model of rat stroke-in-evolution displaying putative penumbral inversion.

BACKGROUND AND PURPOSE: To facilitate reproducible and rigorous study of a tissue zone at risk of encroaching ischemic damage, we propose a new model in which the potentially compromised tissue lies within rather than perifocal to an ischemic locus. The perimeter of the "zone at risk" is defined by a photothrombotically produced cortical lesion in the shape of a toroid (or "ring"). METHODS: The exposed crania of erythrosin B-injected rats were irradiated with a 514.5-nm laser beam, configured as a 5-mm-diameter ring, to yield a ring-shaped lesion caused by photochemically induced platelet occlusion of cortical vasculature. Developing perfusion deficits in the interior region were revealed by carbon black infusion. Tissue damage and infarct volumes were assessed by light and electron microscopy, and blood-brain barrier integrity was assessed with Evans blue dye and horseradish peroxidase as tracers. RESULTS: For rats injected with 17 mg/kg erythrosin B and irradiated for 2 minutes with a ring beam intensity of 0.92 W/cm2 (beam power of 65 mW), carbon black infusion at times up to 4 hours demonstrated a shallow cortical ring lesion encircling a fully patent zone at risk, which by 24 hours evinced an essentially complete perfusion deficit. At times up to 24 hours, the ring lesion was penetrated at the pial surface by distal branches of the middle cerebral and anterior cerebral arteries. Stereotaxically based histopathological assessment showed that by 24 hours the lesion spanned the cortical thickness. Lesion volume increased from 14.5 +/- 8.0 mm3 (mean +/- SD) (n = 8) to 46.2 +/- 15.6 mm3 (n = 8) between 4 and 24 hours after irradiation (P < .01), but the anteroposterior lesion diameter did not change significantly between 4 hours (6.00 +/- 1.03 mm; n = 9) and 24 hours (6.75 +/- 1.15 mm; n = 9). CONCLUSIONS: The present model of slowly developing but inevitable cortical tissue death in a sequestered area should facilitate more precise observations of the evolution of tissue metabolic responses, from the impending onset of ischemia to the threshold of irreversible damage. This system may prove efficient for evaluating treatments intended to salvage a penumbral region.

Characterization of photodynamic actions of rose bengal on cultured cells.

PURPOSE: The authors have previously reported successful photodynamic occlusion of corneal blood vessels using intravenous rose bengal and argon green laser irradiation. To explore the action mechanism of this novel technique of photothrombosis, they examined the photodynamic effect of rose bengal on cultured fibroblasts, smooth muscle cells, and vascular endothelium--the cellular components of blood vessels. METHODS: Five types of cells were exposed to different concentrations of rose bengal and argon green laser irradiation. The irradiated cell areas were analyzed by fluorescence microscopy and fluorometry. Various potential quenchers and proteins were tested for their modulation of the photodynamic action. RESULTS: Upon irradiation with 16 W/cm2 of argon green laser light in conjunction with rose bengal concentrations extending above 1 x 10(-4) M, all cultured cell types showed a dose-dependent photobiologic effect characterized by constriction and detachment of the laser-irradiated cell region from the rest of the cell monolayer. In addition, there was dye photobleaching and development of a blue shift of the fluorescence excitation and emission maxima in the irradiated cell areas. Binding of rose bengal to intracellular components was demonstrated by fluorescence microscopy and by fluorometry showing a red shift of the excitation maximum compared to the maximum in solution. This binding was a prerequisite for expression of the described photobiologic effect, because polymer-conjugated rose bengal (Sensitox II) failed to reproduce it. The addition of native or heat-inactivated bovine serum albumin or catalase decreased this photobiologic effect also owing to dye binding, as indicated by G-75 Sephadex gel filtration chromatography. CONCLUSION: These results indicate that the specific photobiologic effect of monolayer contraction, which simulates the vasoconstriction seen during photothrombosis under argon green irradiation, appears to be caused by the photochemical interaction of rose bengal bound with intracellular components.

Characterization of photochemically induced spinal cord injury in the rat by light and electron microscopy.

This study characterized by light and electron microscopy 49 photochemically induced lesions in adult rat spinal cord at 16 time intervals from 2 days to 17 months after lesioning. Vascular thrombosis, resulting from an intravascular photochemical reaction induced by a rose bengal/laser beam interaction, led within a few days to an extensive area of tissue deterioration. This area, termed the "lesion cavity" in contrast to the "secondary cavity" observed later, was at least 6 mm long and, at the epicenter, extended across most of the spinal cord width and from the dorsal surface to a level near the central canal. The area of spared tissue, 43% of the spinal cord cross-section at 2 days, did not change significantly between 2 and 56 days. Large numbers of macrophages populated the degenerating area by 5 days. This necrotic area was surrounded by a thin peripheral rim of largely intact white matter dorsally and laterally except at the epicenter where the white matter degenerated dorsomedially. In these peripheral regions, demyelination and, by 14 days, remyelination by both oligodendrocytes and Schwann cells (SCs) were evident. By 28 days, far more SCs (and meningeal cells) had entered the dorsal spinal cord, typically at the epicenter where meningeal thickening was most striking, and had migrated farther into the lesion cavity. These SCs and the axons they myelinated remained prominent in dorsal regions for many months, particularly at the epicenter; the proportion of SC to oligodendrocyte myelin diminished away from the epicenter. By 8 weeks, the lesion cavity was considerably diminished in size and thereafter it contained scattered macrophages, SC-myelinated axons, and blood vessels, primarily medially owing to flattening into clefts bilaterally. The cavity was partly bordered by astrocytes whose surfaces toward the lesion cavity were highly irregular and coated with basal lamina. Bare axons, consistently seen by electron microscopy at 5 days to 6 months, were typically ensconced among astrocytes starting at 28 days. Also by this time large, smoothly contoured, empty secondary cavities appeared, usually rostral and caudal to the epicenter; they did not increase in size or number with time. From 28 days to 17 months postlesion they occurred in 68% of the lesioned spinal cords. The secondary cavity border was composed of cells thought to be astrocytes but, surprisingly, the luminal surface was smooth and lacked basal lamina, in contrast to the primary lesion cavity border. Thus, two types of cavities formed after photochemical lesioning. This lesioning technique may provide an appropriate milieu to better understand aspects of the vexing problem of post-traumatic syringomyelia in the human.

Glutamate antagonist MK-801 attenuates incomplete but not complete infarction in thrombotic distal middle cerebral artery occlusion in Wistar rats.

The purpose of this study was to investigate the effects of a non-competitive N-methyl-D-aspartate antagonist, MK-801, on incomplete infarction (selective neuronal necrosis), a zone of which had been found previously in a thrombotic distal middle cerebral artery (MCA) occlusion model in Wistar rats. Male Wistar rats were treated with 1 mg/kg of MK-801 or saline 30 min before MCA occlusion. Laser irradiation with intravenous administration of Rose Bengal dye was used to cause thrombotic distal MCA occlusion. The ipsilateral common carotid artery was occluded permanently and the contralateral carotid artery for 60 min. Head temperature was controlled at 36 degrees C. Cerebral blood flow (CBF) was determined with laser-Doppler flowmetry. Three days after the ischemic insult, brains were perfusion-fixed and volumes of cortical (complete and incomplete) infarction were determined. There were no significant differences in physiological variables or CBF between the two groups. Volumes of complete infarction were equivalent between the two groups (94.9 +/- 15.6 mm3 and 91.6 +/- 14.0 mm3 in the control and MK-801 treated groups, respectively). In MK-801 treated group, the volume of incomplete infarction was reduced by 44% (6.4 +/- 1.7 mm3 vs. 3.6 +/- 2.1 mm3 in control and MK-801 treated groups, respectively, P < 0.05). Although the zone responsive to MK-801 was small in this thrombotic MCA occlusion model, our present study revealed that MK-801 has a beneficial effect on the tissue zone containing selective neuronal alterations (incomplete infarction). Our results support the concept that this drug is effective in the area of less severe ischemia.

Photothrombotic infarction triggers multiple episodes of cortical spreading depression in distant brain regions.

The purposes of this study were to determine whether cortical spreading depression occurs outside of the infarct produced by photothrombotic vascular occlusion, and also the direction of spreading. Focal cerebral thrombotic infarction was produced by irradiating the exposed skull of anesthetized rats with green light (560 nm) following systemic injection of rose bengal dye. At proximal sites (approximately 2 mm anterior to the infarct border), transient, severe hyperemic episodes (THEs) lasting 1-2 min were intermittently recorded. THE frequency was greatest in the first hour and declined over a 3-h period. THEs were accompanied (and usually preceded) by a precipitous rise in [K+]0 (from approximately 3 to > 40 mM) and were associated with increases in local tissue oxygen tension (tPO2). Following the rise in [K+]0, clearance of [K+]0 to its pre-THE baseline preceded baseline recovery of CBF. These data indicate that THEs were reactive to physiologic events resembling cortical spreading depression (CSD), which provoked increased demand for oxygen and blood flow, and which spread from proximal sites to areas more distal (approximately 4 mm) from the rim of the evolving infarct. MK-801 (1 mg/kg, i.v.) inhibited subsequent CSD-like episodes. We conclude that photothrombosis-induced ischemia provoked CSD which was triggered either within the infarct core or in the infarct rim and spread to more distal sites. Whether multiple episodes of CSD during infarct generation are responsible for the remote consequences of focal brain injury remains to be determined.

Molecular genetics of growth and development in Populus. III. A genetic linkage map of a hybrid poplar composed of RFLP, STS, and RAPD markers.

We have evaluated three DNA-based marker types for linkage map construction in Populus: RFLPs detected by Southern blot hybridization, STSs detected by a combination of PCR and RFLP analysis, and RAPDs. The mapping pedigree consists of three generations, with the F1 produced by interspecific hybridization between a P. trichocarpa female and a P. deltoides male. The F2 generation was made by inbreeding to the maximum degree permitted by the dioecious mating system of Populus. The applicability of STSs and RAPDs outside the mapping pedigree has been investigated, showing that these PCR-based marker systems are well-suited to breeding designs involving interspecific hybridization. A Populus genome map (343 markers) has been constructed from a combination of all three types. The length of the Populus genome is estimated to be 2400-2800 cM.