Biomedical subjects
B D Watson
Publications and source records attributed to B D Watson.
Promotion of graft survival by photothrombotic occlusion of corneal neovascularization.
Corneal neovascularization may reduce the success of penetrating keratoplasty. Photothrombosis using intravenous rose bengal and argon laser irradiation has shown promise for occluding corneal vessels. It is therefore conceivable that photothrombosis can improve the graft survival in vascularized corneas. Using intracorneal 7-0 silk sutures as the stimuli, corneal neovascularization was induced in 1 eye each of 19 New Zealand white rabbits. Eleven eyes received photothrombosis. Successful occlusion with subsequent regression was verified by corneal fluorescein angiography. Three were assigned for observation. Six of 8 eyes receiving grafts from an outbred rabbit donor after photothrombosis remained clear during 6.5 to 18.5 weeks of follow-up, while vascularization and opacity occurred in 7 of 8 control eyes without photothrombosis. These results indicate that prior photothrombotic occlusion of corneal vessels can significantly improve graft survival in this experimental model and may have clinical applications.
Morphological consequences of early reperfusion following thrombotic or mechanical occlusion of the rat middle cerebral artery.
The early morphological consequences of recirculation following middle cerebral artery (MCA) occlusion were studied in two rat models. The proximal MCA was occluded for 1 h by either a surgical clip or platelet thrombus; subsequently, 1 h of recirculation was facilitated. Following clip occlusion and recirculation, mild astrocytic swelling, especially around blood vessels, was detected in reperfused cortical and striatal areas. Neuronal changes included slight chromatin clumping and dilation of the rough endoplasmic reticulum. In contrast, severe structural abnormalities were detected following recanalization of the thrombosed MCA segment. Marked astrocytic swelling of cell bodies and perivascular processes with neuropil vacuolation were commonly seen. A heterogeneous pattern of neuronal alterations, including a high frequency of dense shrunken neurons surrounded by swollen astrocytic processes was documented in cortical and striatal regions. Severe neuronal changes were documented in brain regions exhibiting a well-perfused microcirculation. Vascular endothelial cells contained large numbers of pinocytotic vesicles associated with luminal and abluminal surfaces. Pronounced and rapid morphological changes evolve with reperfusion when thrombotic and ischemic events occur simultaneously. The basis for these rapid parenchymal changes following vascular thrombosis may involve acute alterations in cerebral microvascular permeability which exacerbate ischemic consequences.
Acute thrombotic infarction suppresses metabolic activation of ipsilateral somatosensory cortex: evidence for functional diaschisis.
To study the effects of focal infarction on the capacity for functional activation of an ipsilateral somatosensory system remote from the lesion, we produced a small thrombotic infarct in the left frontal pole of male Wistar rats by a photochemical method. Five days later, the awake, restrained rats received tactile stimulation of the large whiskers (vibrissae) of the right side of the face, while a double-label 14C-autoradiographic study of local CMRglc (lCMRglc) and local CBF (lCBF) was performed. Unlesioned and unstimulated animals served as controls. In rats without frontal infarct, vibrissae stimulation led to activation of lCMRglc in the three synaptic relay stations of the barrel-field pathway (ipsilateral trigeminal medullary nucleus, contralateral ventrobasal thalamus, and contralateral barrel-field cortex). The mean increment in lCMRglc was 42% in lamina IV of barrel-field cortex and 49% in ventrobasal thalamus. Normalized lCBF tended to increase in superficial cortical laminae. In unstimulated animals with frontal infarct, lCMRglc was reduced by 20-30% throughout the ipsilateral barrel-field cortex as well as other ipsilateral cortical regions, but not in ventrobasal thalamus or other subcortical areas. In animals with frontal infarct subjected to contralateral vibrissae stimulation, a remarkable suppression of activation was observed throughout the barrel-field cortex so that left-less-than-right hemispheral lCMRglc asymmetry persisted despite stimulation. The ventrobasal thalamus, similarly, failed to increment its lCMRglc with vibrissae stimulation, whereas activation of the trigeminal nucleus was not suppressed. Similar trends were observed in the normalized lCBF data. These observations, which establish that a small frontal infarct is capable of suppressing normal physiological activation in remote ipsilateral brain structures, may have important implications with respect to suppression and recovery of function in human ischemic stroke.
Ischemic injury in the brain. Role of oxygen radical-mediated processes.
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Embolic stroke from a carotid arterial source in the rat: pathology and clinical implications.
We developed a new animal model of stroke which resembles human stroke more closely than existing models. We described the pathology produced in the brain following platelet embolism, previously described only in the retina. The common carotid artery of the rat was irradiated for 6.5 minutes with an argon laser at 514.5 nm after intravenous injection of a photosensitizing agent, rose bengal. A retinal embolus was seen in 1 rat 5 minutes after irradiation. A nonocclusive platelet thrombus was present in the carotid artery 50 minutes after irradiation, with almost all the platelet thrombus being cleared 24 hours later. Acute (1 to 10 days) changes in the brain included 44 small infarcts in 12/13 rats, cortical arterioles occluded with platelets and thickening of small vessels in normotensive rats. Chronic (4 to 12 weeks) changes included lacunes in the brains of normotensive rats and intimal proliferation of smooth muscle in the carotid artery. This is the 1st animal model of (1) stroke with emboli produced in vivo rather than injected into the carotid, (2) intimal proliferation of smooth muscle without invasion of the vessel, and (3) lacunes. This model provides results important to the laboratory study of stroke.
Photothrombosis of corneal neovascularization by intravenous rose bengal and argon laser irradiation.
Management of corneal neovascularization by photocoagulation has been limited and rarely successful. We evaluated the efficacy and safety of the novel technique of photothrombosis to occlude corneal neovascularization. Sixteen rabbit corneas with previous ocular surface wounds that had healed with 360 degrees extensive neovascularization (persistent for 20 months) were used. After an intravenous injection of rose bengal solution (40 mg/kg of body weight [BW]), each vessel on the upper half of the cornea was occluded with a photochemically induced thrombus within ten shots of argon laser irradiation (514.5 nm, 130 mW, 63 microns, 0.2 s); those on the lower half were used as an internal control. Throughout the four-month study period, the treated vessels remained occluded, as evidenced by corneal fluorescein angiography. Corneal clarity was improved after treatment. A single injection of rose bengal at a dose of 8 mg/kg of BW or higher was sufficient for successful photothrombotic occlusion of corneal vessels within one hour of experimentation. Transient elevations of serum urea nitrogen, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and total bilirubin levels and decrease of serum phosphorus level were noted on the first day after injection with 40 mg/kg of BW of rose bengal solution.
A new model of embolic stroke produced by photochemical injury to the carotid artery in the rat.
We report a new model of embolic stroke in the rat, based on endothelial disruption and platelet aggregation in the carotid artery, which leads to distal embolization and focal brain infarction. The common carotid artery was irradiated for 6.5 minutes with the focused beam of an argon laser operated at a wavelength of 514.5 nm with a peak power of 2 W and an average power of 400 mw. Ipsilateral cerebral infarcts, ranging in size from 0.1 to 1.7 mm, were produced by platelet emboli in 12 of 13 rats. A total of 44 infarcts were observed in the 12 rats: 29 in the cortex, 6 in the hippocampus, 5 in the thalamus, and 4 in the basal ganglia. Scanning electron microscopy identified platelet aggregates in the carotid artery and in a deep cortical arteriole 50 minutes after the photochemical lesion. Twenty-four hours after the experiment, scanning electron microscopy of the carotid artery revealed damaged endothelium but few remaining adherent platelets. More intense laser irradiation in 8 rats, leading to carotid occlusion, produced an infarct in only 1. This new model can be used to study the acute and chronic pathological changes in the brain associated with platelet embolism.
Induction of conjunctival transdifferentiation on vascularized corneas by photothrombotic occlusion of corneal neovascularization.
Previous studies have established that conjunctival transdifferentiation (transformation into cornea-like morphology) is inhibited by corneal vascularization. Conversely, occlusion of corneal vessels may induce conjunctival transdifferentiation on vascularized corneas. To test this hypothesis, the corneal epithelia of New Zealand albino rabbits were debrided 3 mm beyond the limbus with n-heptanol. Sixteen corneas healed by conjunctival epithelium, with vascularization persisting for 20 months, were used in this study. Photochemically induced occlusion of the corneal vessels was achieved by intravenous administration of rose bengal-saline solution (40 mg/kg body weight) with subsequent argon laser irradiation of the vessels (514.5 nm, 130 mW, 63 micron and 0.2 sec). The treated vessels remained occluded in an 18-week study, as confirmed by corneal fluorescein angiography. Corneal clarity and epithelial integrity were improved after treatment. Goblet cell loss and morphologic transformation into a cornea-like epithelium were verified by flat-mount preparations, histology, impression cytology, and immunofluorescence studies using a mucin-specific monoclonal antibody. These results indicate that conjunctival transdifferentiation can be induced on vascularized corneas after occlusion of corneal vessels by photothrombosis.
Hyperglycemia increases infarct size in collaterally perfused but not end-arterial vascular territories.
Hyperglycemia exacerbates neuronal injury in the setting of reversible brain ischemia, but its effect on focal thrombotic infarction has been less extensively characterized. We investigated this problem in two rat models of focal vascular occlusion. In Model I, the right middle cerebral artery (MCA) was exposed via a subtemporal craniotomy in halothane- and nitrous oxide-anesthetized Wistar rats and was occluded photochemically by irradiation with an argon ion laser following the intravenous administration of the photosensitizing dye rose bengal. Permanent MCA occlusion was combined with temporary bilateral common carotid artery ligation. In Model II, similarly anesthetized Sprague-Dawley rats were subjected to permanent photochemical occlusion of the right MCA without common carotid occlusion. In both models, rats were food deprived for 24 h and were administered varying amounts of 50% dextrose (or saline) 15 min prior to vascular occlusion to produce a spectrum of plasma glucose values, ranging from 5 to 44 mumol/ml. Brains were examined histologically 7 days following vascular occlusion, and computer-assisted planimetry was used to compute infarct volumes. In Model I, the volume of neocortical infarction ranged from 30.3 to 108.4 mm3 and exhibited a strong linear correlation with increasing preischemic plasma glucose values (r = 0.70). In contrast, the size of the smaller striatal infarct in this model was not correlated with plasma glucose level. In Model II, there was a prominent striatal infarct, ranging in volume from 14.4 to 96.4 mm3, while neocortical infarction occurred inconstantly. As in Model I, striatal infarct volume in Model II showed no correlation with plasma glucose level.(ABSTRACT TRUNCATED AT 250 WORDS)
Photothrombotic occlusion of rat middle cerebral artery: histopathological and hemodynamic sequelae of acute recanalization.
The histopathological and hemodynamic consequences of photochemically induced middle cerebral artery (MCA) thrombosis and recanalization were studied in the rat. Recanalization of the thrombosed MCA segment was achieved by the topical application of nimodipine at 1 h following photochemically induced occlusion. Pathological consequences of permanent and temporary occlusion were compared by morphometric procedures 7 days following thrombus formation. Rats with permanent thrombosis exhibited consistent infarction of both striatum and cortex. MCA recanalization at 1 h was associated with a significant reduction in total infarct volume. In recanalized rats, small cortical infarcts, confined to the peripheral MCA territory, were observed in only three of six rats. In contrast, a mixed pattern of infarction and ischemic cell damage was documented throughout the striatum in all rats. Local CBF (1CBF), measured autoradiographically, was significantly reduced in the MCA territory following 1 h of MCA occlusion, especially within the striatum. At 1 h after recanalization, 1CBF recovered within the previously ischemic brain regions to greater than 50% of control. Perfusion deficits were detected by carbon black infusion within focal areas of the striatum following reperfusion. Thus, cortical neurons appear to tolerate 1 h of MCA occlusion in this model. In contrast, reperfusion following 1 h of photochemically induced MCA occlusion gives rise to selective injury to the striatum.
Middle cerebral artery thrombosis: acute blood-brain barrier consequences.
The effect of middle cerebral artery (MCA) thrombosis on the integrity of the blood-brain barrier (BBB) was studied in rats using horseradish peroxidase (HRP). Endothelial injury with subsequent platelet thrombosis was produced by means of a rose bengal-sensitized photochemical reaction, facilitated by irradiating the right proximal MCA segment with the focused beam of an argon laser. At 15 minutes following thrombosis formation, diffuse leakage of HRP was observed bilaterally within cortical and subcortical brain areas. Peroxidase extravasation was most dense within the territory of the occluded artery including neocortical areas and dorso-lateral striatum. Contralaterally, a similar distribution was observed but with less intense HRP leakage. Ultrastructural studies demonstrated an increase in permeability to HRP within arterioles, venules and capillaries. At these sites, the vascular endothelium contained HRP-filled pinocytotic vesicles and tubular profiles. Although less intense, bilateral HRP leakage was also observed following MCA stenosis or femoral artery occlusion. Endothelial-platelet interactions at the site of vascular injury may be responsible for releasing substances or neurohumoral factors which contribute to the acute opening of the BBB.
Photochemically stimulated blood-borne factors induce blood-brain barrier alterations in rats.
We have tested the hypothesis that blood-borne substances released from a site of vascular thrombosis can lead to acute alterations in the blood-brain barrier. The right common carotid artery of rats was photothrombosed using a dye/light insult. Rats were given the photosensitizing dye rose bengal and irradiated for 4 minutes with an argon laser beam focused onto the exposed common carotid artery. During the irradiation, 3 ml of blood was taken from the right external carotid artery. After 10 minutes, the blood was infused into the external carotid artery of a recipient rat that had received horseradish peroxidase. Fifteen minutes after blood infusion, bilateral peroxidase extravasation was noted within cortical and subcortical areas of recipient rats, being most intense ipsilaterally. Ultrastructural studies demonstrated peroxidase reaction product within numerous endothelial vesicles of arteriolar segments. Infusion of blood from control rats did not produce similar changes. Thus, photoinduced vascular thrombosis of a large feeding artery leads to the formation of blood-borne factors that acutely alter cerebral vascular permeability.
Amelioration of experimental lipid keratopathy by photochemically induced thrombosis of feeder vessels.
The photochemical interaction between intravenously injected rose bengal dye and 514.5-nm argon laser irradiation was employed to initiate permanent thrombotic occlusions in the corneal neovasculature of rabbit eyes with experimentally induced lipid keratopathy. This photothrombotic procedure did not produce corneal edema or polymorphonuclear leukocyte infiltration, as was found in previous studies employing laser-induced photocoagulation. Thus, by means of the new technique, we avoided the formation of additional neovascularization and lipid deposition. Vascular occlusion by photothrombosis, effected with 8.5 times less incident intensity and 27.5 times less total light exposure than with photocoagulation, yielded an average reduction of corneal cholesterol content of 36% as opposed to an increase of 24% found with previous argon laser photocoagulation.
Photochemically induced cerebral infarction. I. Early microvascular alterations.
Cerebral ischemia leading to infarction was produced in rats by intravascular thrombosis induced by a photochemical reaction between systemically injected rose bengal and green light (560 nm) transmitted through the intact skull for a 2-min period. At 2 or 15 min following photochemical sensitization, animals were perfusion-fixed for scanning (SEM) and transmission (TEM) electron microscopic analyses of the cerebral vasculature. At 2 minutes, ultrastructural examination of cortical regions destined to undergo infarction revealed numerous platelet aggregates within both pial and intraparenchymal vessels. Platelets close to the endothelial walls were routinely degranulated with pseudopodia. Endothelial cells were frequently swollen and contained dilated mitochondria and granular endoplasmic reticulum. The endothelial luminal membrane structure was shown by high-power TEM to be focally damaged. If brain temperature was reduced by 4 degrees C during the photochemical sensitization period, the platelet response was inhibited without interfering with other ultrastructural changes. These results are consistent with the hypothesis that photochemically induced endothelial alterations stimulate platelet activation and implicate abnormal endothelial function as a primary event in the pathogenesis of photochemically induced cerebral infarction.
Photochemically induced cerebral infarction. II. Edema and blood-brain barrier disruption.
Alterations in the blood-brain barrier to proteins, and regional water and electrolyte content were documented in a rat model of photochemically induced small-vessel thrombosis leading to infarction. Horseradish peroxidase (HRP) or Evans blue was given immediately following a 2-min photochemical sensitization period. At 5 min following irradiation, multifocal sites of peroxidase extravasation were noted within the irradiated area. Ultrastructural examination revealed endothelial cells filled with HRP which in some cases extended into the basal lamina and extracellular spaces. At 15 min, protein leakage was more pronounced within the irradiated zone and reaction product was also apparent within the subarachnoid and perivascular spaces of brain regions remote from the site of irradiation. Widespread staining on the surface of the irradiated hemisphere was apparent in rats perfused 8 h following Evans blue infusion. Water content increased significantly by 15 min within the irradiated zone but not in brain regions remote from this site. Although vasogenic edema is an early event in this stroke model, increases in water content are restricted to the irreversibly damaged site. In contrast, protein tracer escaping from microvessels coursing within the irradiated zone was widely distributed. These findings implicate endothelial barrier dysfunction in the genesis of tissue injury in this model. Morphological evidence for the capability of macromolecules to escape from a site of evolving infarction and to migrate to distances remote from the area of primary microvascular damage is also discussed.
Argon laser-induced arterial photothrombosis. Characterization and possible application to therapy of arteriovenous malformations.
The common carotid, femoral, and middle cerebral arteries in the rat have been occluded thrombotically by means of a rose bengal dye-sensitized photochemical reaction initiated in vascular endothelium by the 514.5-nm beam of an argon laser, focused for maximum excitation efficiency of the photosensitizer according to a derived criterion. The total energy required for vessel occlusion was approximately 1 joule (J) for the middle cerebral artery and 140 to 180 J for the femoral and carotid arteries. At energy fluences (energy deposited per unit area) of 3.5 kJ/sq cm for the middle cerebral artery and 35 kJ/sq cm for the larger arteries, occlusion was observed within 3 minutes. The middle cerebral artery thrombus consisted entirely of aggregated platelets; in the larger arteries the thrombi were composed of platelet aggregates and groups of red blood cells interspersed within a matrix of coagulum. Vessels irradiated similarly in the absence of rose bengal dye displayed no morphological or functional damage. Because the photochemical reaction is mediated by electronic-state transitions, the process of photothrombosis (as opposed to photocoagulation) can be initiated in vessels with high flow rates without the requirement of increased temperature. The photothrombotic technique may be useful in the treatment of arteriovenous malformations owing to its significant enhancement of the efficiency and permanency of vessel occlusion.
Photochemically induced graded spinal cord infarction. Behavioral, electrophysiological, and morphological correlates.
Neurological and morphological outcome was evaluated in a rat model of graded spinal cord infarction initiated by a photochemical reaction. In this model, light-dye interactions induce primary microvascular stasis, resulting in consistent patterns of tissue necrosis. Four groups of rats underwent photoinduction times ranging from 30 seconds to 10 minutes. Neurological and electrophysiological functions were assessed starting 1 week after irradiation and continuing for 8 weeks. A functional neurological score was obtained by combining results from sensory and motor tasks, and electrophysiological function was evaluated from the somatosensory evoked potential recordings. In rats irradiated for short periods (30 seconds and 1 minute) mild behavioral deficits were documented. In contrast, electrical conduction was suppressed acutely in both groups; this recovered by 8 weeks to baseline or near baseline in the 30-second group but not in the 1-minute group. In rats irradiated for longer periods (5 and 10 minutes), severe behavioral and conduction abnormalities were detected at both the subacute and chronic testing periods. Although no significant difference in behavior was documented between the 5- and 10-minute groups acutely, the rats with 5-minute photoinduction time demonstrated a significant improvement in behavior over time whereas the group with 10-minute photoinduction time showed no improvement. A severe conduction block was present in both animal groups during the course of the study. Histopathological examination combined with morphometric measurements of the lesion area in cross section revealed four different degrees of spinal cord necrosis which correlated significantly with photoinduction times and neurological scores at 8 weeks. Reproducible degrees of ischemic damage to spinal cord parenchyma following primary microvascular occlusion result in a predictable sequence of behavioral and functional abnormalities, which in some cases recover with time.