Bilateral Clamping Of Carotid Arteries Research Articles

THE INFLUENCE OF ACUTE CEREBRAL BLOOD CIRCULATION DISORDERS IN THE POOL OF CAROTID ARTERIES ON THE RNA CONTENT IN THE CELLS OF THE LYMPHOID POPULATION OF THE RATS’ THYMUS OF DIFFERENT AGE GROUPS

The aim of the work – to find out the age-related features of the response of RNA cells ofthe lymphoid population of the thymus to an acute disturbance of cerebral blood flow inthe pool of carotid arteries in six- and nine-month-old rats.Materials and methods. Acute cerebrovascular accident was simulated in 6- and9-month-old white non-linear laboratory rats by bilateral carotid artery clamping for 20min. followed by reperfusion. The thymus was taken on the 12th day of the postischemicperiod, fixed in Buen’s solution, after standard histological processing it was embedded inparaffin, and serial sections with a thickness of 5 μm were made. After deparaffinization,rehydration, and staining with hallocyanine- chromium alum according to Einarson,histological sections were analyzed in a digital image analysis system VIDAS-386(Kontron Electronik, Germany) with a fluorescent microscope AXIOSKOP (Zeiss,Germany). Total RNA content was expressed in units of optical density (EOP per 1 mm2). The results. In the cells of the lymphoid population of all structural and functional zones ofthe thymus of control rats of both age groups, an increase in RNA content was establishedin the direction from immature to more mature classes (p<0.05). This regularity persistsin the post-ischemic period. In the cells of the lymphoid population of the subcapsular anddeep cortical zones of the thymus, the RNA content significantly prevails in six-month-oldrats, in intralobular perivascular spaces – in nine-month-old rats, and in the medullaryzone there are no age differences. Postischemic changes in RNA content in six-month-oldrats in the subcapsular zone and intralobular perivascular spaces of the thymus consistin its decrease in immature cell classes and its increase in mature small thymocytes(р<0.05); in the deep cortex – in growth in all classes of thymocytes (p<0.05). In ninemonth-old animals, the differences were in the increase in RNA content in the subcapsularzone and in less mature middle thymocytes, in the deep cortex – in a decrease in RNAcontent in all classes, except small thymocytes, in intralobular perivascular spaces – ina decrease in RNA content in all classes of thymocytes (p<0.05 for all cases). In themedullary zone of animals of both age groups, changes are unidirectional (decrease inRNA content, p<0.05).Conclusion. Age differences in the nature of postischemic changes in the content of RNAin thymocytes are present in all structural and functional zones of the gland, with theexception of the medullary, in which the changes were unidirectional in both age groups.

AGE CHARACTERISTICS OF CHANGES IN THE LYMPHOID POPULATION STRUCTURE OF THE RATS’ THYMUS AFTER ACUTE DISTURBANCE OF CEREBRAL BLOOD FLOW IN THE BASIN OF CAROTID ARTERIES

Abstract. Today, the most common causes of disability and mortality on the planet include acute disorders of cerebral blood circulation, the spectrum of which is dominated by ischemic lesions. Advances in neuroimmunology and neuroimmunopathology have made it possible to attribute cerebral blood circulation disorders to immune- dependent diseases of the nervous system, therefore, the emphasis in the search of new approaches to the eff ectiveness increase of stroke therapy has shifted from the correction of local changes in the brain itself to the reaction of the immune system organs, as a possible target of pharmacological eff ects, that will make it possible to reduce the secondary death of neurons as a result of infl ammation.The purpose of the work. to investigate the age-specifi c changes in the structure of the lymphoid population of the thymus after acute disruption of cerebral blood fl ow in the basin of the carotid arteries in six- and ninemonth-old rats.Material and methods. Acute cerebrovascular accident was simulated in 6- and 9-month-old white nonlinear laboratory rats by bilateral carotid artery clamping for 20 min. followed by reperfusion. The thymus was taken on the 12th day of the postischemic period, it was fi xed in Buen’s solution, after standard histological processing, serial sections with a thickness of 5 μm were made, stained with hematoxylin- eosin. In the subcapsular, deep cortical zones, intralobular perivascular spaces and the medullary zone of the thymus, the structure of the lymphoid population (density of diff erent classes of thymocytes per 1 mm2) was studied in the digital image analysis system VIDAS-386 (Kontron Electronik, Germany) with a fl uorescent microscope AXIOSKOP (Zeiss, Germany) in automatic mode.The results. In the structure of the lymphoid population of all structural and functional zones of the thymus, small lymphocytes predominate. Age-related diff erences in the structure of the lymphoid population of the thymus of the control groups consist of lower indicators of the density of medium and small thymocytes in all investigated zones of the gland of animals of the older age group, with the exception of the subcapsular, in which the density of small thymocytes is higher in nine-month-old rats. On the 12th day after simulation of ischemia- reperfusion of the brain injury the number of destructive thymocytes and thymocytes with the signs of apoptosis increases in the subcapsular and deep cortical zones of the thymus of six-month-old rats, but in nine-month-old rats in all zones of the gland, except the brain, the density of destructive and apoptotic cells decreases.Conclusions. Ischemic- reperfusion injury of the brain modifi es the structure of the lymphoid population of the thymus of rats, and the nature of these modifi cations is determined by age and structural and functional zones of the gland.

Influence of Fenpropathrin on Memory and Movement in Mice After Transient Incomplete Cerebral Ischemia

Fenpropathrin, a synthetic pyrethroid widely used as an insecticide, is known to affect locomotion and memory in mammals. It is possible that exposure to pyrethroids may occur in an elderly population where transient ischemic attacks are a higher risk for occurrence with consequent changes in memory and control of movement. Thus, the aim of this study was to determine whether bilateral clamping of carotid arteries (BCCA), a model for ischemia, together with fenpropathrin affected memory in tests such as the passive avoidance task and fresh spatial memory in a Y-maze, as well as movement activity and movement coordination on a rotarod in mice. BCCA together with fenpropathrin significantly reduced latency in a passive avoidance task compared to controls. There were no significant differences among the groups with respect to the Y-maze, movement activity, or movement coordination. In conclusion, fenpropathrin needs to be used with caution in the presence of an elderly population at risk for ischemia, as there appears to be evidence of some memory loss in mice.

Levemopamil reduces spatial learning deficit following transient occlusion of common carotid arteries in normotensive rats.

The effect of the calcium channel blocker and 5-HT2 antagonist levemopamil on spatial learning impairment of rats subjected to transient bilateral clamping of carotid arteries (BCCA) was investigated. In addition, the acute effect of BCCA on local cerebral blood flow was measured in the presence and absence of levemopamil in a separate group of rats. Pretreatment with levemopamil prevented the BCCA-induced increase in escape latency during the test trials and ameliorated the BCCA-induced decrease in spatial bias during a probe trial. Under these experimental conditions, local cerebral blood flow fell to near ischaemic values in all mid- and forebrain regions during occlusion of the carotid arteries. However, pretreatment with levemopamil affected the BCCA-induced blood flow changes only in one brain area of 34 investigated. The present data suggest that pretreatment with levemopamil reduces impairment in spatial behaviour and that this effect seems not related to the compound's cerebral vasodilatory action, but to direct neuronal mechanisms.

Expression of pannexin2 protein in healthy and ischemized brain of adult rats

The expression pattern of the pannexin2 protein (Px2) in healthy and ischemized brains of adult rats was investigated. A polyclonal antibody for rat Px2 was generated in chicken and purified for affinity. This antibody was used to study by Western blot, Enzyme-Linked Immunosorbent Assay, and immunohistochemistry, the expression pattern of Px2 in healthy brain of adult rats and in the hippocampus of rats submitted to bilateral clamping of carotid arteries for 20 min, followed by different times of reperfusion (I/R) (8 h, 24 h, 48 h, 72 h, 14 days and 30 days). Immunohistochemical studies visualized the wide and complex expression pattern of Px2 in the healthy brain. All Px2 + positive cells were neurons which also showed no puncta on their cellular membranes. Both pyramidal cells and interneurons, the majority of which were positive to parvalbumin, were stained in healthy hippocampus. The number of Px2 interneurons in the hippocampus showed a progressive reduction at successive time intervals after I/R, with a negative peak of about −40% after 72 h from I/R. Interneurons which were positive for both Px2 and parvalbumin, represented about the 85% of all parvalbumin cells stained in the hippocampus. This percentage rested grossly unmodified at different time intervals after I/R in spite of the progressive neuronal depletion. Concomitantly, an intense astrogliosis occurred in the hippocampus. Most of the astroglial cells expressed de novo and for a transient time (from 24 h to 14 days from I/R), Px2. Primary co-cultures of hippocampal neurons and astrocytes were submitted to transient ischemia-like injury. This set of experiments further confirmed the in vivo results by showing that Px2 is de novo and transiently expressed in astroglial cells following a transient ischemia-like injury. These results suggested the expression of Px2 in the astrocytes may be induced either from injured neurons or by biochemical pathways internal to the astrocyte itself. In conclusion, our results showed the transient expression of Px2 in astrocytes of reactive gliosis occurring in the hippocampus following I/R injury. We hypothesize that Px2 expression in astrocytes following an ischemic insult is principally involved in the formation of hemichannels for the release of signaling molecules devoted to influence the cellular metabolism and the redox status of the surrounding environment.

2-Deoxyglucose enhances epileptic tolerance evoked by transient incomplete brain ischemia in mice

The aim of the study was to assess the influence of chronic treatment with a non-metabolisable glucose analogue, 2-deoxyglucose (2-DG) at a 150 mg/kg dose on long-term epileptic tolerance (ET) evoked by 30 min bilateral carotid artery clamping (BCCA) in mice. The effects of protein synthesis inhibition with cycloheximide (CHX), given in three daily doses of 2.5 mg/kg starting either 1 day before (peri-insult regimen) or 1 day after the priming insult (post-insult regimen), on ET development was also studied. Seizures were induced 14 days after BCCA with 3.5 mg/kg of bicuculline; this dose (CD 97) evokes convulsions in 97% of normal untreated mice. BCCA resulted in decreased mortality, prolonged latency to the onset of generalised convulsions and decreased overall seizure score. CHX given in the post-insult regimen did not influence, while the peri-insult regimen abolished, all signs of BCCA-evoked ET. 2-DG treatment of sham-operated animals resulted in a moderate but significant decrease in mortality rate and a tendency toward a lower seizure score. BCCA combined with 2-DG treatment resulted in a marked decrease in mortality rate, as well as reduction in all indicators of seizure susceptibility. CHX abolished the antiepileptic effects of BCCA alone, as well as BCCA combined with 2-DG, while it did not influence the 2-DG-related decrease in mortality. We conclude that the development of BCCA-induced epileptic tolerance, as well as unmasking antiepileptic effects of 2-DG by BCCA, is dependent on protein synthesis.

Levemopamil injection after cerebral oligemia reduces spatial memory deficits in rats

Transient reduction of cerebral blood flow to oligemic levels as produced by bilateral clamping of carotid arteries (BCCA) in pentobarbital anesthetized Wistar rats leads to spatial orientation deficiencies in a water maze test 8–10 days after surgery. These deficiencies are more pronounced in 4-month-old than in 6-week-old animals. Levemopamil [(2S)-2-isopropyl-5-(methylphenethylamino)-2-phenylvaleronitrile hydrochloride], a Ca 2+ channel blocker and 5-HT 2 antagonist, prevents the deficiencies in groups of animals of both ages, even when administered 24 h after the transient vessel occlusion. Levemopamil did not influence the maze performance of sham-operated control rats. Levemopamil, therefore, appears to modulate mechanisms that are altered specifically either by, or as a consequence of, the BCCA procedure. Levemopamil did not influence the altered GABA or ACh content in different vulnerable brain structures following BCCA, showing that the substance acts via additional mechanisms affected by the BCCA procedure.

Effects of bilateral clamping of carotid arteries on hippocampal kindling in rats

Moderate reduction of cerebral blood flow by bilateral clamping of carotid arteries (BCCA) in pentobarbital anaesthetized Wistar rats induces decreased PO 2 and temperature values in vulnerable brain structures such as hippocampus and frontal cortex during the acute phase of clamping. Up to two weeks chronic increased GABA contents in hippocampus, substantia nigra and frontal cortex could be observed. During this time the development of hippocampal kindling was difficult. These results demonstrated an acute and chronic influence of GABA on most vulnerable brain structures which, however, do not lead to ischemic impacts as histological analyses demonstrate.

Decreased susceptibility to seizures induced by bicuculline after transient bilateral clamping of the carotid arteries in rats

Rats were exposed for 24 min to bilateral clamping of the common carotid arteries (BCCA) in pentobarbital anaesthesia. 14 days later the animals were subjected to subcutaneous injection of (+)-bicuculline (3 or 4 mg/kg). A significantly decreased susceptibility to bicuculline-induced seizures could be observed in BCCA treated rats compared with sham operated controls. It is suggested that BCCA treatment protects animals against status epilepticus and lethal toxicity produced by bicuculline. Electrographic recordings of the BCCA animals revealed no ictal activity within 1 h after bicuculline injection. An analysis of the GABA content showed a significant increase in the hippocampus (HPC), frontal cortex (FCX), parietal cortex and substantia nigra in BCCA animals compared with controls. It is therefore possible that an increase in GABA content postsynaptically counteracts the GABAA antagonistic effect of bicuculline in BCCA animals thus preventing the normal seizure inducing effect of this substance.

Postischemic insulin reduces spatial learning deficit following transient forebrain ischemia in rats.

We investigated the ability of postischemic insulin administration to modify the structural and neurobehavioral consequences of cerebral ischemia in rats. Forebrain ischemia was induced in fed rats by combining controlled systemic hypotension with bilateral carotid artery clamping for 10 1/2 minutes. Following clamp release, one group of six rats [corrected] was given insulin (2 IU/kg s.c. b.i.d.) for 1 week. An ischemic-control group of five rats [corrected] received no postischemic treatment. A sham-ischemia group of rats was used as a behavioral control. Throughout the recovery period until sacrifice, the drinking water of all rats was supplemented with 25% glucose. Rats were trained on two water maze place navigation tasks 1-2 months after ischemia. Escape latencies and swim patterns were recorded. Performance in the insulin-treated group was better than that in the ischemic-control group (p less than 0.05) on both tasks and did not differ significantly from that of the sham-ischemia group. Improvement in behavior correlated with a significant reduction in CA1 hippocampal necrosis in the insulin-treated group (p less than 0.05). Our findings demonstrate that postischemic treatment with insulin improves neurobehavioral performance in addition to lessening ischemic neuronal necrosis.

Circulating catecholamines modulate ischemic brain damage.

In search of factors influencing the outcome of an ischemic insult, we induced 10 min of forebrain ischemia in rats and assessed neuronal necrosis by quantitative histopathology after 1 week of recovery. Procedures for inducing ischemia included bilateral carotid artery clamping and reduction of blood pressure to 40-50 mm Hg by bleeding. To facilitate rapid lowering of blood pressure, a ganglionic blocker, trimethaphan (TMP), was administered at the onset of ischemia. Omission of the ganglionic blocker proved to markedly ameliorate neuronal damage. Similarly favorable effects were obtained when a mixture of adrenaline and noradrenaline (1 microgram kg-1 min-1 each) was infused during the early recirculation period in animals previously given TMP. Infusion of noradrenaline alone also ameliorated the damage, though the efficacy was somewhat less. The results suggest that catecholamines, released as a response to stress, ameliorate ischemic brain damage.

Forebrain ischemia in the rat. Relation between duration of ischemia, use of adjunctive ganglionic blockade and long-term recovery.

The relation between duration of ischemia, use of adjunctive ganglionic blockade and long-term recovery was studied in a rat model giving reversible subtotal forebrain ischemia. Ischemia was induced by bilateral carotid artery clamping and controlled hemorrhage to a mean arterial pressure of 50 mm Hg in animals artificially ventilated under 70% N2O. After variable lengths of time, the clamps were removed and the drawn blood was reinfused. In some animals, the ganglion blocker Arfonad was given (group A+) on induction of ischemia to facilitate hypotension. There was a strict dose-response relationship between duration of ischemia and mortality. Mortality was higher among animals not given Arfonad (group A-; 37% after 10 min of ischemia and 100% after 13 min) than in group A+ (about 20% after 12-13 min of ischemia, 50% after 15 min and 80% after 19 min). In group A+ more than half of the animals died later than 24 h after ischemia. All of them were hyperexcitable and 12% died during witnessed epileptic fits. Group A- animals regularly died within the first 24 h, with no indication of central nervous system involvement. Less blood had to be drawn to attain hypotension (mean arterial pressure 50 mm Hg) in group A+ (1.5 +/- 0.3 ml/100 g b.w.) than in group A- (2.5 +/- 0.2 ml/100 g b.w.). Group A+ also had less "washout" acidosis 5 min after reinfusion of the shed blood than group A- (15 min of ischemia: pH 7.24 +/- 0.07 v 6.96 +/- 0.06).(ABSTRACT TRUNCATED AT 250 WORDS)

Models for studying long-term recovery following forebrain ischemia in the rat. 2. A 2-vessel occlusion model.

A model is described in which transient ischemia is induced in rats anaesthetized with N2O:O2 (70:30) by bilateral carotid artery clamping combined with a lowering of mean arterial blood pressure to 50 mm Hg, the latter being achieved by bleeding, or by bleeding supplemented with administration of trimetaphan or phentolamine. By the use of intubation, muscle paralysis with suxamethonium chloride, and insertion of tail arterial and venous catheters, it was possible to induce reversible ischemia for long-term recovery studies. Autoradiographic measurements of local CBF showed that the procedure reduced CBF in neocortical areas, hippocampus, and caudoputamen to near-zero values, flow rates in a number of subcortical areas being variable. Administration of trimethaphane or phentolamine did not affect ischemic and postischemic flow rates, nor did they alter recovery of EEG and sensory-evoked responses, but trimetaphan blunted the changes in plasma concentrations of adrenaline and noradrenaline. Recovery experiments showed that 10 min of ischemia gave rise to clear signs of permanent brain damage, with a small number of animals developing postischemic seizures that led to the death of the animals in status epilepticus. After 15 min of ischemia, such alterations were more pronounced, and the majority of animals died. It is concluded that the short revival times noted are explained by the fact that the model induces near-complete ischemia, and that recovery following forebrain ischemia is critically dependent on residual flow rates during the period of ischemia.

Brain lactic acidosis and ischemic cell damage: A topographic study with high-resolution light microscopy of early recovery in a rat model of severe incomplete ischemia

Transient severe incomplete ischemia was induced in rats by a combination of bilateral carotid artery clamping and hypovolemic hypotension. Production of lactic acid in the ischemic brain was modified by preischemic administration of glucose or saline. After 30 min of ischemia and 5 or 90 min of recirculation, the animals were fixed by perfusion. High-resolution light microscopy based on whole hemisphere plastic sections revealed that the model produces a highly predictable ischemia in the telencephalon, with a more inconstant injury in the diencephalon, rostral brain stem, and cerebellum. The extent of injury correlates well with studies of local cerebral blood flow in the same model. The present study largely confirmed the opinion, based on the earlier study of the frontoparietal cortex, that the neuronal injury is predominantly of the 'pale' type, although fair amounts of 'dark' injury also appeared with predilection to the pyriform cortex, hippocampus, and occasionally the cerebellum. Excessive tissue lactic acidosis due to glucose pretreatment aggravated both types of neuronal injury. It was also accompanied by marked astrocytic edema as well as capillary obstruction in the group with long recirculation. A novel type of ischemic tissue change emerged, consisting of osmiophilic granules and whorls probably derived from damaged cell membranes.

Brain lactic acidosis and ischemic cell damage: 1. Biochemistry and neurophysiology.

This study explores the influence of severe lactic acidosis in the ischemic rat brain on postischemic recovery of the tissue energy state and neurophysiological parameters. Severe incomplete brain ischemia (cerebral blood flow below 5% of normal) was induced by bilateral carotid artery clamping combined with hypovolemic hypotension. We varied the production of lactate in the tissue by manipulating the blood glucose concentrations. A 30-min period of incomplete ischemia induced in food-deprived animals caused lactate to accumulate to 15-16 mumol g-1 in cortical tissue. Upon recirculation these animals showed: (1) a considerable recovery of the cortical energy state as evaluated from the tissue concentrations of phosphocreatine, ATP, ADP, and AMP; and (2) return of spontaneous electrocortical activity as well as of somatosensory evoked response (SER). In contrast, administration of glucose to food-deprived animals prior to ischemia caused an increase in tissue lactate concentration to about 35 mumol g-1. These animals did not recover energy balance in the tissue and neurophysiological functions did not return. In other experiments the production of lactate during 30 min of complete compression ischemia was increased from about 12 mumol g-1 (normoglycemic animals) to 20-30 mumol g-1 by preischemic hyperglycemia and, in separate animals, combined hypercapnia. The recovery of the cortical energy state upon recirculation was significantly poorer in hyperglycemic animals. It is concluded that a high degree of tissue lactic acidosis during brain ischemia impairs postischemic recovery and that different degrees of tissue lactic acidosis may explain why severe incomplete ischemia, in certain experimental models, is more deleterious than complete brain ischemia.

Influence of complete and pronounced incomplete cerebral ischemia and subsequent recirculation on cortical concentrations of oxidized and reduced glutathione in the rat.

Abstract: The influence of complete and pronounced incomplete cerebral ischemia on cortical concentrations of reduced (GSH) and oxidized (GSSG) glutathione was studied in lightly anaesthetized (70% N2 O) rats. GSH was extracted with HCl‐methanol‐perchloric acid and GSSG with trichloroacetic acid in the presence of N‐ethylmaleimide and measured fluorometrically, giving normal concentrations in cortical tissue of about 2 and 0.01 μmol.g−1 respectively. Reversible complete ischemia was induced by increasing the intracranial pressure to above the systolic blood pressure by infusing mock CSF into the cisterna magna. Reversible pronounced incomplete ischemia was induced by bilateral carotid artery clamping combined with hypovolemic hypotension. Whether complete or incomplete, a 30‐min ischemic period caused a similar decrease in cortical GSH concentration (to about 90% of control) without any concomitant accumulation of GSSG in the tissue (or in CSF). Prolongation of the ischemic period (complete ischemia) to maximally 120 min caused an almost linear decrease of the tissue glutathione concentration to 45% of the preischemic value. During subsequent recirculation following a 30 min period of either complete or pronounced incomplete ischemia, there was a further decrease in cortical GSH concentrations without a reciprocal increase in GSSG concentrations. Lipid peroxidation (verified by determination of malondialdehyde production) induced in brain cortical tissue in vitro caused oxidation of tissue GSH with accumulation of GSSG. As the observed decrease in GSH during brain ischemia in vivo was not accompanied by any reciprocal increase in GSSG the results fail to support the hypothesis that peroxidative damage occurs during or following brain ischemia. The finding of an unchanged GSSG concentration does, however, not exclude the possibility of an increased turnover rate in the glutathione reductase reaction. It is concluded that the observed decrease in tissue GSH concentration mainly reflects a decrease in the glutathione pool size, due to an imbalance between breakdown and synthesis secondary to tissue energy failure.

Distribution of renal cortical blood flow during hemorrhagic hypotension in conscious dogs.

AbstractThe effect of hemorrhagic hypotension on distribution of renal cortical blood flow was investigated in 9 conscious dogs. 6 platinum electrodes were chronically implanted in inner and outer renal cortex and local cortical blood flow measured by hydrogen clearance. Urine was collected through a catheter implanted in the split bladder and total renal blood flow f RBF) determined by PAH clearance. In anesthetized dogs control blood flow averaged 3.72 ± 0.74 ml/min‐g in outer cortex and 3.60 ± 0.53 ml/rnin g in inner cortex. Local blood flow in conscious dogs fell to 30–60 % of control in the course of 11 days after implantation of the electrodes, though RBF was almost constant and filtering glomeruli were demonstrated in the small fibrotic zone (20–50 μ) close to the electrode tip. No change in local flow was found by induction of Nembutal anesthesia. Bleeding of conscious dogs 1–3 days after implantation, to mean arterial pressure 50 mm Hg, reduced local blood flow to 10–30 % of prebleeding control. The local blood flow in outer and inner cortex decreased near proportionately. Typically, flow at all electrode sites in outer and inner cortex showed a similar fall. A marked redistribution of cortical blood flow during hemorrhagic hypotension was never observed. Bilateral clamping of carotid arteries at normal and at low arterial pressures did not change outer or inner cortical blood flow.