Bilateral Carotid Ligation Research Articles

Sequential analysis of subacute and chronic neuronal, astrocytic and microglial alterations after transient global ischemia in rats.

Recent experimental investigations have emphasized the importance of assessing both acute and chronic histopathological changes occurring after cerebral ischemia. The purpose of this study was to evaluate the temporal profile of neuronal, astrocytic and microglial alterations within vulnerable regions (striatum and CA1 sector of hippocampus) following transient global ischemia. Anesthetized Wistar rats underwent 10 min of normothermic (37 degrees C) ischemia induced by bilateral carotid ligations plus hypotension (45-50 mm Hg) and were allowed to survive for periods ranging from 1 to 10 weeks (n=4-6/group) prior to quantitative histopathological analysis. Adjacent sections were examined by hematoxylin-and-eosin histopathology, immunostaining for glial fibrillary acidic protein, and B4-isolectin immunochemistry for microglia. In the striatum, normal-neuron counts were first decreased significantly at 2 weeks after the ischemic insult. Neuronal loss was associated with the proliferation of reactive microglia, which peaked at 1 week. By contrast, reactive astrocytosis displayed a more protracted pattern, with peak activation at 2 weeks. In the CA1 hippocampus, a decreased number of normal neurons was seen at 1 week post ischemia, together with a significant increase in immunoreactive microglia at that time; the latter normalized after 2 weeks. Reactive astrocytes in the CA1 hippocampus were significantly increased at 1-2 weeks after ischemia. In a subgroup of severely injured animals, foci of frank striatal infarction were associated with early and severe microglial and astrocytic proliferation at week 4 or later. Finally, cerebrovascular changes included endothelial disruption within affected areas. These observations document a subacute and chronic sequence of cellular responses following brief periods of global ischemia, involving both neurons, glia and vascular endothelium.

Effects of diaspirin cross-linked haemoglobin on post-traumatic cerebral perfusion pressure and blood flow in a rodent model of diffuse brain injury

Diaspirin cross-linked haemoglobin (DCLHb) is a new oxygen carrying blood substitute with vasoactive properties. Vasoactive properties may be mediated via high affinity binding of nitric oxide by the haem moiety. Using a rodent model of head injury combined with ischaemia, we studied the effects of DCLHb on cerebral blood flow (CBF) and intracranial pressure (ICP). Twenty anaesthetized rats were allocated randomly to receive treatment with DCLHb 400 mg kg-1 i.v. or placebo (oncotically matched plasma protein substitute 4.5% i.v.). To produce diffusely increased ICP, after a severe weight drop injury, all animals underwent a 30-min period of bilateral carotid ligation combined with a period of induced hypotension. After reperfusion, DCLHb or placebo was infused and the animals instrumented for measurement of intraventricular ICP and CBF in the region of the sensorimotor cortex using the hydrogen clearance technique. Mean arterial pressure (MAP), ICP, cerebral perfusion pressure (CPP) (CPP = MAP - ICP) and CBF were measured 4 h after injury in all animals. DCLHb significantly reduced ICP from mean 13 (SEM 2) to 3 (1) mm Hg (P < 0.001), increased CPP from 52 (8) to 95 (6) mm Hg (P < 0.001) and increased CBF from 21 (2) to 29 (2) ml 100 g-1 min-1 (P = 0.032). We conclude that DCLHb improved CPP without a reduction in CBF in a rodent model of post-traumatic brain swelling.

C007 High potassium (K) diets reduce mortality and brain injury after bilateral carotid ligation

C007 High potassium (K) diets reduce mortality and brain injury after bilateral carotid ligation

Chronic cerebral hypoperfusion by permanent internal carotid ligation produces learning impairment without brain damage in rats

To investigate the influence of cerebral hypoperfusion on learning behaviours, we developed a novel rat cerebral hypoperfusion model, in which the bilateral internal carotid arteries were permanently ligated to reduce the cerebral blood flow, and examined its behavioural and histopathological consequences in comparison to those occurring after bilateral common carotid ligation. In the Morris water maze task, rats with common carotid ligation exhibited a learning deficit, whereas rats with internal carotid ligation exhibited normal learning. Both models exhibited significant learning impairments in the eight-arm radial maze task, although the impairment was less severe in internal carotid-ligated rats than in common carotid-ligated rats. The cerebral blood flow of rats with common carotid ligation was reduced significantly both two and 10 days after ligation, and was still below normal three months after ligation. A milder, but significant reduction in the cerebral blood flow was observed in internal carotid-ligated rats. Shrinkage of the optic nerves and a circadian activity rhythm desynchronized to the light/dark cycle were exhibited by the rats with common carotid ligation, whereas these parameters remained unaffected in the rats with internal carotid ligation, suggesting that permanent ligation of common carotid arteries but not internal carotid arteries impairs visual functions. The main pathological changes observed in the brain following common carotid ligation were rarefaction and gliosis of the white matter and neuronal loss in the hippocampal CA1 region. On the other hand, the rats with internal carotid ligation had no significant brain damage. Chronic treatment with idebenone (1.5 and 15 mg/kg/day), a cerebral energy metabolism enhancer, over a three-month period, commencing five days after ligation, ameliorated the impairment of water maze learning in rats with common carotid ligation. The treatment also significantly improved the learning impairment in the radial maze task of internal carotid-ligated rats. Idebenone had no effect on the histopathological changes that followed cerebral hypoperfusion. It is concluded that cerebral hypoperfusion induced by permanent internal carotid ligation impairs the working memory without causing pathological damage to the brain tissues and the visual system, and the learning impairment can be ameliorated by a cerebral energy metabolism enhancer. These findings have the clinical implication that a reduction in blood flow may be an important factor that causes or exacerbates cognitive decline in dementias.

Selective impairment of response to acetylcholine after ischemia/reperfusion in mice.

We previously reported that the endothelium-dependent dilation of pial arterioles by either topical acetylcholine (ACh) or bradykinin (BK) was markedly inhibited after 10 minutes of near total ischemia after bilateral carotid occlusion. The present study tests the responses after 10 minutes of reperfusion and investigates the effect of either oxygen or oxygen radical scavengers on the results. Mice were subjected to bilateral carotid ligation or sham ligation. Pial arteriolar diameters were monitored by an image-splitting technique at a craniotomy site. In separate studies, the responses to topically suffused ACh, BK, or sodium nitroprusside (SNP) were tested before ischemia. After 10 minutes of ischemia and 10 minutes of reperfusion, the response was assessed again. Sham-operated mice were observed in each study. Cerebral blood flow was continuously monitored with a laser-Doppler technique. Additional separate studies were conducted as follows: presence of superoxide dismutase plus catalase during ischemia and reperfusion, or increase in the inspired oxygen (arterial oxygen) and oxygen in suffusate. The response to ACh was significantly impaired after 10 minutes of reperfusion. The responses to BK and SNP were unaffected. Radical scavengers failed to influence the impaired response to ACh. Elevations of arterial and suffusate oxygen levels to over 300 mm Hg failed to prevent the impairment. After 10 minutes of reperfusion, a selective impairment of the response to ACh remains. The response to another endothelium-dependent dilator, BK, recovered, and the response to endothelium-independent SNP was unaffected. Because neither radical scavengers nor oxygen altered the outcome with respect to ACh, I suggest that neither radical generation nor hypoxia accounts for the selective impairment of dilation by ACh. Rather, I hypothesize that reduced shear during ischemia diminishes the ability of the endothelium to synthesize and/or release the endothelium-derived relaxing factor for ACh. I hypothesize further that this impaired release or synthesis persists throughout the 10-minute period of reperfusion.

Effect of triiodothyronine on graft function in a model of heart transplantation

Brain death is associated with neuroendocrine changes that result in impaired metabolism, reduced myocardial energy stores, and deteriorating cardiac function. As a result of these changes, a substantial number of normal human hearts are not considered suitable for transplantation. In the hope of preventing these complications and stabilizing the condition of cardiac donors, we compared the function of transplanted hearts from brain-dead rats that received triiodothyronine (T3) (n = 6) with that of hearts from a group that received a placebo (n = 5). This experiment was designed to be both blinded and randomized. Brain death was achieved by bilateral carotid ligation and inflation of an intracranial balloon. Triiodothyronine or placebo was administered in a blinded, randomized fashion. The brain-dead donors were then supported with conventional techniques for 2 hours after which time heterotopic transplantation was performed using hypothermic preservation and a working heart model. Hemodynamics of the transplanted hearts were assessed 48 hours postoperatively. The hearts from donors that had been pretreated with T3 were found to have a significantly higher (p < 0.005) peak left ventricular pressure than the hearts from the placebo-treated group (137 +/- 17 mm Hg versus 115 +/- 15 mm Hg). Left ventricular end-diastolic pressure was significantly lower (p < 0.01) in the T3-treated group (5.2 +/- 2.2 mm Hg) compared with the placebo-treated group (6.9 +/- 0.5 mm Hg). There was also a significantly higher (p = 0.03) maximal first derivative of left ventricular pressure in the T3-treated group compared with the placebo-treated group (4,876 +/- 1,348 mm Hg/s versus 3,344 +/- 1,016 mm Hg/s). Finally, the cardiac output in the group given T3 was 93 +/- 16 mL/min compared with 61 +/- 22 mL/min in the group given the placebo (p < 0.01). Hearts from brain-dead rats that had received T3 before transplantation showed improved postoperative function. The experimental design of predonation brain death, cold immersion storage, and transplantation in a working heart model should make these data more relevant clinically than those previously reported.

Comparative evaluation of acute cerebral vasospasm by the microsphere and the angiography techniques

Experimental subarachnoid haemorrhage (SAH) induces an acute transient cerebral vasospasm. The goal of this study was to compare angiography with iterative measurements of regional cerebral blood flow (rCBF) by the microsphere technique for tracking acute cerebral vasospasm after SAH. Cerebral vasospasm was induced in anaesthetised rabbits by injecting 1 ml of fresh blood in the cisterna magna. In a first experiment, the diameter of the basilar artery was measured by repeated angiograms over 60 min. In a second experiment, rCBF was measured over 60 min by the radioactive microspheres method without and with bilateral ligation of the carotid artery. Without carotid ligation, despite a profound transient vasospasm of the basilar artery, rCBF was unchanged in the cerebellum and cerebrum and was not statistically decreased in the brain stem. However, with bilateral carotid ligation, rCBF dramatically decreased at 5 and 15 min after haemorrhage. At 30 min, despite a persistent 50% decrease in the basilar cross-sectional area, rCBF was no longer different from the control group. Thus in a model of acute vasospasm of the basilar artery, rCBF evaluation by the microsphere technique parallels the cerebral vasospasm evaluated by angiography only when both carotid arteries are ligated.

Ascorbic acid during cerebral ischemia in newborn piglets.

We measured ascorbic acid (reduced and oxidized) in brain, CSF and blood, before, during and after cerebral ischemia in newborn piglets. Bilateral carotid ligation induced a 54% decrease in cerebral blood flow (p < 0.01) and a 43% decrease in the cerebral metabolic rate of oxygen (p < 0.01). After ischemia and reperfusion, we obtained a 60% decrease (p < 0.01) in total brain ascorbic acid content. CSF ascorbic acid increased during reperfusion: +60% at 30 min (p < 0.001) and +160% at 120 min (p < 0.05). Blood ascorbic acid content did not change. These changes and the absence of massive oxidation of ascorbic acid in brain tissue suggest release of ascorbic acid by the brain during ischemia.

Neuroprotective effect of human osteogenic protein-1 in a rat model of cerebral hypoxia/ischemia

Possible neuroprotective actions of osteogenic protein-1 (OP-1) were evaluated in a rat model of cerebral hypoxia/ischemia. Intraperitoneal injection of 50μg of OP-1 prior to bilateral carotid ligation and transient hypoxia in 12-day-old rats reduced cerebral infarct area from 44.8 ± 3.3% in vehicle-injected controls to 29 ± 4.9%. Treatment of 14-day-old rats with 20μg of OP-1 1 h after hypoxia reduced mortality from 45% to 13%. OP-1 may represent a novel class of neuroprotective agents.

Effects of MK-801 and ganglioside GM1 on postischemic prostanoid release and hippocampal lesion in gerbil brain

In this study Mongolian gerbils were submitted to a normothermic bilateral carotid ligation lasting 5 min. A noncompetitive antagonist of NMDA receptors, MK-801, 0.8 mg/kg, was injected i.p. 30 min before ischemia, or the ganglioside GM1, 30 mg/kg, was given i.p. for 3 days, twice a day. The morphology of the hippocampal CA1 neurones and the brain content of cyclooxygenase metabolites of arachidonic acid: prostaglandin 6-keto PGF1 alpha and thromboxane Tx B2 were studied. Untreated ischemia induced the accumulation in brain of the 6-keto PGF1 alpha and Tx B2 immunoreactive materials, and resulted in a lesion of 70% of CA1 neurones. In the MK-801- and GM1-pretreated groups the postischemic levels of Tx B2 were significantly decreased. However MK-801 and GM1 did not prevent damage to the CA 1 neurones in gerbils normothermic after ischemia, whereas a partial neuroprotection was observed in hypothermic, MK-801 treated gerbils. The results of this study indicate that NMDA receptors may participate in the mechanism of postischemic release of eicosanoids in brain. They also confirm a potential modulatory role of gangliosides. These results are discussed in terms of the involvement of cyclooxygenase metabolites of arachidonic acid in the mechanism of a selective delayed neuronal damage to the hippocampus CA1 after ischemia.

Cardiovascular responses and central catecholamines in streptozocin-diabetic rats

Brain catecholamine levels, spinal cord levels of the norepinephrine metabolite methoxy-hydroxy-phenylglycol (MHPG), and heart rate were measured in nondiabetic and streptozocin-diabetic rats after sham surgery or bilateral carotid ligation. Although carotid ligation increased heart rate in both diabetic and nondiabetic rats, in diabetic animals the response did not differ from the response to sham surgery. Carotid ligation increased epinephrine concentrations in the medulla/pons of diabetic animals but was not associated with alterations in other central catecholamines. In all diabetic rats spinal ratios of MHPG/norepinephrine (an index of noradrenergic activity) were higher than in nondiabetics, and the change in heart rate (post-surgical-pre-surgical rate) correlated inversely with hypothalamic dopamine (R = -0.60). In sham-operated diabetic rats there were high inverse correlations of the change in heart rate with medullary epinephrine and of pre- and post-surgical heart rate with spinal MHPG/NE (R = -0.87 to -0.95). Central catecholamines and heart rate were not correlated in nondiabetic animals and correlated only weakly when nondiabetic and diabetic animals were pooled. Correlations in diabetic animals were usually abolished or reduced by carotid ligation. These findings suggest a link between central catecholamines and heart rate in diabetic rats subjected to surgical stress. Whether catecholaminergic neurons contribute to abnormal chronotropic responses in diabetic rats or respond to stimuli that affect both heart rate and neural function remains to be determined.

High-Cervical Spinal Cord Electrical Stimulation in Brain Low Perfusion Syndromes: Experimental Basis and Preliminary Clinical Report

Previous studies of our group showed that C1-C2 spinal cord stimulation increases carotid and brain blood flow in normal conditions in the goat and dog and it has a beneficial vasomotor effect in a model of vasospasm in the rat. For further clinical application it seemed rational to investigate the possible vascular changes mediated by this technique in experimental brain infarction. To this aim, 45 New Zealand rabbits were used. Brain infarction was produced by bilateral carotid ligation in 15, unilateral microcoagulation of the middle cerebral artery in 15 and by microcoagulation of the vertebral artery at the craniocervical junction in the other 15. One week later, following daily clinical scoring and cortical and posterior fossa blood flow readings by laser Doppler, a period of 120 min of right C1-C2 spinal cord electric stimulation was performed. A mean of 27% increase in previous blood flow recordings was obtained at the right hemisphere and a mean of 32% in the posterior fossa. This procedure was used in 10 patients presenting with various cerebral low perfusion syndromes. Though not constant, an increase in alertness, retention, speech, emotional lability and performance in skilled acts was achieved. No MR changes were observed, though SPECT readings showed an increase in blood flow in the penumbral perilesional area.

The role of histamine in brain oedema formation.

The effects of histamine on the cerebral endothelial cells were studied. To determine if the extent of brain oedema formation could be reduced with histamine receptor antagonists, mepyramine (H1-receptor blocker), metiamide, cimetidine and ranitidine (H2-receptor antagonists) were administered at a dose of 5 mg/kg body weight 4, 2 and 0 h before the onset of experimental pneumothorax induced in newborn piglets. Mepyramine and ranitidine given 2 h before the induction of EBP prevented the accumulation of water, sodium and albumin in samples taken from the parietal cortex. In other experiments, carried out on Sprague-Dawley rats of CFY strain after permanent bilateral common carotid ligation (BCCL), the accumulation of water and sodium in the ischemic brain tissue could also be prevented in a dose dependent manner by intraperitoneal injections of ranitidine given 30 min before the surgery. Taken together, these results provide pharmacological evidence for the involvement of histamine receptors in the pathogenesis of brain oedema. Consequently, the use of histamine receptor blockers both in the prevention and in the treatment of brain oedema can be recommended.

Nitric oxide production during focal cerebral ischemia in rats.

Nitric oxide has been implicated as a mediator of glutamate excitotoxicity in primary neuronal cultures. A number of indicators of brain nitric oxide production (nitric and cyclic guanosine monophosphate [cGMP] concentrations and nitric oxide synthase activity) were examined after bilateral carotid ligation and right middle cerebral artery occlusion in adult rats. Brain nitrite was significantly increased in the right versus left cortex 5, 10, and 20 minutes after middle cerebral artery occlusion (P < .05), with a return to baseline at 60 minutes. There were no significant changes in cerebellar concentrations. Cortical levels of cGMP were increased at 10, 20, and 60 minutes after occlusion, with significant right-to-left differences (P < .05). Cerebellar concentrations of cGMP were also increased but without significant side-to-side differences. Nitric oxide synthase activity increased approximately 10-fold from baseline 10 minutes after occlusion in the right cortex but decreased markedly by 60 minutes from its peak at 10 minutes. The right-to-left difference in nitric oxide synthase activity was significant at 20 minutes (P < .05). Pretreatment of rats with NG-nitro-L-arginine, a nitric oxide synthase inhibitor, abolished the rise in nitrite and cGMP. These results suggest that a sharp transient increase in the activity of nitric oxide synthase occurs during the first hour of cerebral ischemia, which leads to a burst in nitric oxide production and activation of guanylate cyclase.

Posthypoxic treatment with felbamate is neuroprotective in a rat model of hypoxia‐ischemia

Felbamate, a novel dicarbamate anticonvulsant that blocks the glycine site of the N-methyl-D-aspartate receptor and protects the hippocampal slice from hypoxic damage, shows remarkably low toxicity in animals and in humans. Since most treatment of human cerebral ischemia will have to be delivered after the insult, we investigated the neuroprotective potency of post hoc felbamate in rat pups with bilateral carotid ligations exposed to an atmosphere of 6.5% O2 for 1 hour. Brain temperature was unaffected by surgery, hypoxia, or felbamate. Neuroprotection was greatest at 300 mg/kg, less effective at 200 and 400 mg/kg, and ineffective at 100 mg/kg. Post hoc felbamate (300 mg/kg) reduced the volume of infarction from 67% +/- 7% of neocortex in unmedicated rats to 32% +/- 8%, 51% +/- 12%, 38% +/- 19%, and 53% +/- 10% when given 0, 1, 2, and 4 hours after hypoxic exposure, respectively. By 6 hours, post hoc protection was no longer significant. Delayed neuronal necrosis in hippocampal granule cells was reduced from 156 +/- 33 neurons to 12 +/- 7 (0 hours, p < 0.01) and 37 +/- 17 (1 hour, p < 0.05). These effects were obtained at plasma concentrations (60 to 120 mg/ml) that have occasionally been reached without serious toxicity in human anticonvulsant trials. These data suggest that, in this animal model, felbamate given after a hypoxic-ischemic insult is effective in reducing cerebral infarction and extremely effective in preventing delayed neuronal necrosis, but that the window of opportunity for post hoc treatment is only 1 to 4 hours.

Felbamate reduces hypoxic-ischemic brain damage in vivo

The neuroprotective effects of felbamate were tested in a model of incomplete cerebral ischemia and hypoxia in 7-day-old rat pups. Felbamate pretreatment (300 mg/kg) reduced the surface of infarcted cortex following bilateral carotid ligation, by 42–49% compared to saline and dimethylsulfoxide (DMSO) controls, respectively. The number of necrotic neurons in the d dentate gyrus was reduced by 77% over both DMSO controls and saline controls. These results suggest that felbamate deserves further evaluation for its therapeutic potential in hypoxia-ischemia.

Chapter 5: Adrenalectomy aggravates ischemic brain edema in female Sprague-Dawley rats with carotid arteries ligated

The effect of adrenalectomy has been investigated in a model of global cerebral ischemia. After bilateral carotid ligation the mortality rate was increased in adrenalectomized rats, and this effect was prevented by glucocorticoid pre-treatment. Adrenalectomy accelerated the appearance of the symptoms of cerebral ischemia, resulting in a moderate aggravation of brain edema and in a significant decrease in the concentration of high-energy phosphate esters. Our findings support the view that endogenous glucocorticoids may play a role in the amelioration of ischemic brain injuries in rats.

Claudication on mastication following bilateral external carotid ligation

Ligation of both external carotid arteries to control epistaxis can produce a decrease in the blood supply of the muscles of mastication sufficient to cause pain typical of claudication while chewing. We report 3 cases of this complication. We advocate that this procedure be used only in special circumstances.

The NMDA antagonist MK-801 improved metabolic recovery after 10 minutes global cerebral ischaemia in rats measured with 31 phosphorous magnetic resonance spectroscopy

The blockade of postsynaptic receptors for excitatory amino acids is a promising new field for the possible treatment of cerebral ischaemia. The most important receptor seems to be the N-methyl-D-aspartate (NMDA) subtype of the glutamate receptors and MK-801 is a potent non-competitive antagonist to the NMDA receptor. 31P NMR Spectroscopy was used to measure the recovery of intracellular pH and the high energy phosphates Phosphocreatine (PCr) and ATP after ten minutes of temporary global cerebral ischaemia in the rat. Cerebral ischaemia was obtained by combining bilateral carotid ligation and systemic hypotension (2 vessel occlusion model). Two intervention groups with intravenous injection of MK-801 in doses of 0.25 mg/kg and 0.5 mg/kg 15 minutes before onset of ischaemia were compared to a control group. Both intervention groups showed a more rapid recovery of PCr and ATP than the control group, but there were no significant differences for intracellular pH.

Trophic changes in the arteries at the base of the rat brain in response to bilateral common carotid ligation.

A rat model was developed to examine changes in the posterior circle of Willis and the basilar and intracranial vertebral arteries after bilateral common carotid ligation. This procedure produced a major redistribution of blood to the head, with increased flow through the vertebral and basilar arteries. Changes in the vertebral, basilar, posterior communicating and proximal segments of the posterior cerebral arteries and neck vessels were assessed by postmortem barium sulfate arteriography and by histology of the middle portion of the basilar arteries serially at four days, and one, two, four and 15 weeks post-ligation. The changes noted were basilar and vertebral artery tortuosity, enlargement, and duplication of the vertebro-basilar junction. By 15 weeks, these intracranial vascular changes had largely regressed toward normal, commensurate with the appearance of multiple collateral vessels which were scattered throughout the soft tissues of the neck and shunted the original ligation sites. A mechanism that could explain these trophic vessel changes in response to increased blood flow is discussed. Some possible correlates of these findings with several brain vascular diseases are presented.