Unilateral Common Carotid Artery Ligation Research Articles

The effect of age on susceptibility to brain damage in a model of global hemispheric hypoxia-ischemia

Stroke occurs in all age groups, ranging from the newborn to the elderly. The immature brain is generally believed to be more resistant to the damaging effects of cerebrovascular compromise compared to the more mature brain. However, recent experiments suggest that the correlation between brain damage and age is not linear. To determine the effects of age and development on hypoxic-ischemic brain damage, we developed a model whereby rats of increasing age received identical cerebrovascular insults, and assessed neuropathologic outcome. Male Wistar rats of 1, 3, 6, and 9 weeks and 6 months underwent unilateral common carotid artery ligation and exposure to 12% oxygen for 35 min. Animals were all spontaneously breathing under light halothane anesthesia (0.5%). Core temperatures were maintained at 37°C. Blood pressures were monitored via indwelling carotid artery catheters on the side ipsilateral to the carotid artery ligation. Cerebral blood flow was assessed in separate groups utilizing Laser Doppler flowmetry. Physiologic monitoring revealed that under these experimental conditions, mean arterial blood pressure and cerebral blood flow decreased to the same extent in each of the age groups, verifying that all animals experienced an identical insult. Neuropathologic assessment at 7 days of recovery showed that brain damage was most severe in the 1 and 3 week old animals followed by those that were 6 months. The 6 and 9 week old groups had significantly less injury than the other 3 age groups. Hippocampal damage was most severe in the 3 week and 6 month old rats compared to all other age groups. Our findings contrast previously held beliefs regarding the enhanced tolerance of the immature brain to hypoxic-ischemic damage and demonstrates that, in a physiologically controlled in vivo model of hemispheric global ischemia, (1) the immature brain is, in fact, less resistant to hypoxic-ischemic brain damage than its adult counterpart, (2) the brain damaging effects of hypoxic-ischemia are age dependent, but do not increase linearly with adbancing age and development, and (3) the intermediate age groups are more tolerant to hypoxic-ischemic brain injury than either very young or more mature ages.

EFFECTS OF MILD HYPOTHERMIA ON AMINO ACID EFFLUX DURING HYPOXIA-ISCHEMIA(H-I) IN THE IMMATURE RAT. † 2281

We have previously shown that following 3 hrs of H-I at 31, 34, or 37°C; 0, 40, and 90% of 7-day rat pups respectively displayed brain damage(Ped Res.29:366,1991). This effect is only partially explained by a preservation of high-energy phosphates in the 31°C group, since ATP is equally depleted in the 34 and 37°C groups (Can.J.Neurol.Sci. 21:S48, 1994). To further investigate the mechanisms underlying the neuroprotective effect of mild hypothermia, in vivo microdialysis was utilized to determine hippocampal amino acid (AA) efflux during H-I. 7-day rats underwent unilateral common carotid artery ligation and exposure to hypoxia in 8% oxygen for 3 hours at either 31, 34, or 37°C. Microdialysis probes (CMA 11) were placed in the ipsilateral CA1 region of the hippocampus, and dialysate collected for 20 min. at 1 hr prior to, and at 60, 120, and 180 min during H-I. Samples were immediately analyzed for alanine, glutamine, glutamate, glycine, taurine, serine, and GABA by high-performance liquid chromatography with electrochemical detection. Prior to H-I, there were no differences between groups in extracellular AA concentrations. During H-I, the concentrations of all AA, except glutamine, rose significantly above control values (p<0.001) only in the 37°C group. In the 34°C group, only alanine increased above control (p<0.01), whereas the other AA remained within control limits. No increases in AA levels were seen in the 31°C group. Thus, 1) The mechanism of protection by mild hypothermia is temperature dependant, 2) Mild hypothermia of 34°C exerts its protective effect by suppressing excitatory AA release, and 3) excess alanine in the extracellular fluid may be a sensitive indicator of brain injury in the immature rat.(Supported by the Heart & Stroke Foundation of Saskatchewan).

THE EFFECT OF AGE ON SUSCEPTIBILITY TO HYPOXIC-ISCHEMIC (H-I) BRAIN DAMAGE.▴ 2282

The immature brain is believed to be more resistant to the damaging effects of H-I compared to the more mature brain. However, recent experiments suggest that the correlation between brain damage and age is not linear. To determine the effects of age and development on H-I brain damage, we developed a model whereby rats of increasing age received identical cerebrovascular insults, and assessed neuropathologic outcome. Male Wistar rats of 1, 3, 6, and 9 weeks and 6 months underwent unilateral common carotid artery ligation and exposure to 12% oxygen for 35 minutes. Animals were all spontaneously breathing under light halothane anesthesia (0.5%). Core temperatures were maintained at 37°C. Blood pressures were monitored via indwelling carotid or femoral catheters. Cerebral blood flow was assessed in separate groups utilizing Laser Doppler flowmetry. Physiologic monitoring revealed that under these experimental conditions, mean arterial blood pressure and cerebral blood flow decreased to the same extent in each of the age groups, verifying that all animals experienced an identical insult. Neuropathologic assessment at 7 days of recovery showed that brain damage was most severe in the 1 and 3 week old animals followed by those that were 6 months. The 6 and 9 week old groups had significantly less injury than the other 3 age groups. Hippocampal damage was most severe in the 3 week and 6 month old rats compared to all other age groups. Our findings contrast previously held beliefs regarding the enhanced tolerance of the immature brain to H-I damage and demonstrates that, in a physiologically controlled in vivo model of hemispheric global ischemia, 1) the immature brain is less resistant to H-I brain damage than it's adult counterpart, 2) the brain damaging effects of H-I are age dependent, but do not increase linearly with advancing age and development, and 3) intermediate age groups are most tolerant to H-I brain injury.

Paradoxical mitochondrial oxidation in perinatal hypoxic-ischemic brain damage

Measurements of cytoplasmic and mitochondrial markers of the oxidation-reduction (redox) state of brain tissue were conducted in a perinatal animal model of cerebral hypoxia-ischemia to ascertain underlying biochemical mechanisms whereby ischemia (reduced oxygen and substrate supply) causes brain damage. Seven-day postnatal rats underwent unilateral common carotid artery ligation followed by exposure to 8% oxygen at 37°C for 3 h. During the course of hypoxia-ischemia, the rat pups were quick frozen in liquid nitrogen and their brains processed for the enzymatic, fluorometric measurement of cerebral metabolites necessary for the calculation of intracellular pH and cytoplasmic and mitochondrial redox states. The results showed an early mitochondrial reduction followed by re-oxidation during the course of hypoxia-ischemia. The oxidation reflected a partial depletion in accumulated reducing equivalents and coincides temporally with the duration of hypoxia-ischemia required to convert selective neuronal necrosis into cerebral infarction. The findings suggest that perinatal cerebral hypoxia-ischemia is characterized more by a limitation of substrate than of oxygen supply to the brain, which may explain why glucose supplementation of the immature animal improves neuropathologic outcome, in contrast to adults.

Sevoflurane improves neurological outcome after incomplete cerebral ischaemia in rats

We have studied the effects of sevoflurane on neurological outcome in a rat model of incomplete cerebral ischaemia. After institutional approval, 30 non-fasted male Sprague-Dawley rats (455-555 g) were anaesthetized, the trachea intubated and the lungs ventilated mechanically with isoflurane and 30% oxygen in air. Catheters were inserted into the right femoral artery, both femoral veins and into the right jugular vein for measurement of arterial pressure, drug administration and blood sampling. At completion of surgery, isoflurane was discontinued and the rats were allowed an equilibration period of 30 min according to the following regimens: group 1 (n = 10) received 70% nitrous oxide in oxygen and fentanyl (bolus 10 micrograms kg-1 i.v.; infusion 25 micrograms kg-1 h-1); group 2 (n = 10) received 1.98 vol% sevoflurane in oxygen and air (FIO2 0.3); group 3 (n = 10) received 1.98 vol% sevoflurane in oxygen and air (FIO2 0.3) and 40% glucose (6 ml kg-1 i.p.) 30 min before ischaemia. Ischaemia was produced by combined unilateral common carotid artery ligation and haemorrhagic hypotension to 35 mm Hg for 30 min. Temperature, arterial blood-gas variables and arterial pH were maintained within the physiological range. Plasma glucose concentration was measured before, during and after ischaemia. Neurological deficit was evaluated for 3 days after ischaemia. Neurological outcome was better in sevoflurane anaesthetized animals, regardless of the plasma glucose concentration, compared with nitrous oxide-fentanyl controls. This indicates that differences in plasma glucose concentrations do not account for the cerebral protection seen with sevoflurane.

Temporal evolution of neuropathologic changes in an immature rat model of cerebral hypoxia: a light microscopic study.

The sequential evolution of neuropathologic changes was studied in an immature model of cerebral hypoxia-ischemia. According, 7-day postnatal rats were subjected to unilateral common carotid artery ligation combined with 2 h of hypoxia (breathing in 8% oxygen) and their brains were examined by light microscopy at recovery intervals ranging from 0 to 3 weeks. Immediately following hypoxia, a large area with a pale staining border was noted occupying most of the cerebral hemisphere ipsilateral (IL) to the occluded common carotid artery; in approximately half of the brains the dorsomedial cortex of the contralateral (CL) hemisphere was also involved. Most neurons in the pale area had nuclei containing a coarse granular condensation of chromatin. Within a few hours, the majority of neurons in the IL hemisphere had developed pyknotic nuclei and clear or eosinophilic perikarya. After 24 h these changes had evolved in the majority of brains into coagulation necrosis (infarction) in the IL hemisphere and foci of selective neuronal necrosis in the CL cortex. Within a few days infarcts became partially cavitated, and by 3 weeks a smooth-walled cystic infarct had developed. Activated microglia/macrophages and reactive astrocytes were first seen at 4 and 24 h, respectively. No parenchymal neutrophilic infiltrate was seen at any time point.

Effect of status epilepticus on hypoxic-ischemic brain damage in the immature rat.

Seven-day postnatal rats were subjected to unilateral common carotid artery ligation, 3 h after which they were subjected to hypoxia with 8% oxygen at 37 degrees C for 2 h. Thereafter, they received multiple s.c. injection(s) of bicuculline (6 mg/kg) adequate to produce behaviorally apparent seizures lasting greater than 1 h (status epilepticus). Repeated episodes of status epilepticus at 2, 6, and 12 h of recovery from hypoxia-ischemia (HI) produced a mortality rate of 53%. Among the survivors, there was no statistically significant difference in the extent of brain damage between convulsing and non-convulsing HI controls, analyzed at 30 d of age. Histopathologic examination for acute lesions also indicated no difference in the severity of brain damage between dead and surviving rat pups subjected to status epilepticus, indicating that mortality was not related to the severity of prior HI brain damage. Those immature rats that died during status epilepticus exhibited lower blood glucose concentrations (1.75 +/- 0.35 mmol/L) compared with surviving, convulsing animals (4.25 +/- 0.51 mmol/L; p = 0.016). Glucose supplementation (0.1 mL of 50% glucose) early during status epilepticus improved survival and significantly prolonged seizure activity (90 +/- 14 min) compared with non-glucose treated, convulsing littermates (47 +/- 10 min; p = 0.02). Glucose supplementation did not increase the extent of brain damage despite improved survival and increased duration of seizure activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Magnetic Resonance Imaging of Brain Edema in the Neonatal Rat: A Comparison of Short and Long Term Hypoxia-ischemia

Diffusion-weighted and transversal relaxation time (T2)-weighted magnetic resonance imaging were used to study the relationship between the duration of hypoxia-ischemia [unilateral common carotid artery (CCA) ligation and exposure to 8% oxygen] and the in vivo visualization of brain edema in 7-d-old rats. After CCA ligation, 35 animals were divided into five groups according to the length of exposure to 8% oxygen: no exposure (n = 9), 15 min (n = 12), 30 min (n = 5), and 1 h (n = 9) exposure; six animals served as controls. Diffusion weighted images were acquired 2 h after the hypoxic-ischemic insult, sequential T2 weighted images were recorded for up to 7 d and the outcome was documented by histologic examination at 21 d. The apparent diffusion coefficient of water in the ipsilateral cortex was significantly decreased in all animals recovering from prolonged hypoxic-ischemic insult (30 min and longer), whereas this was the case in only 40% of animals exposed to 15 min of hypoxia. Moreover, T2 prolongation of brain tissue occurred only in the former group. These results indicate transient and reversible alterations of physiologic water compartmentation for short term hypoxia-ischemia, but irreversible edema formation for long term hypoxia-ischemia. They support the hypothesis that the duration of hypoxia-ischemia determines whether a vasogenic edema and infarction follows the initial cytotoxic edema.

Carbon Dioxide Protects the Perinatal Brain From Hypoxic-Ischemic Damage: An Experimental Study in the Immature Rat

Clinical investigations suggest that premature infants who require mechanical ventilation from respiratory distress syndrome are at increased risk for periventricular leukomalacia if hypocapnia occurs during respiratory management. The question remains as to the contribution of hypocapnia to hypoxic-ischemic brain damage and whether or not hypercapnia is neuroprotective. Seven-day postnatal rats underwent unilateral common carotid artery ligation followed thereafter by exposure to systemic hypoxia with 8% oxygen (O2) combined with either 0, 3, 6, or 9% carbon dioxide (CO2) for 2.5 hours at 37 degrees C. Survivors underwent neuropathologic examination at 30 days of postnatal age, and their brains were categorized as follows: 0 = normal; 1 = mild atrophy; 2 = moderate atrophy; 3 = atrophy with cystic cavitation < 3 mm; 4 = cystic cavitation > 3 mm of the cerebral hemisphere ipsilateral to the carotid artery ligation. The width of the ipsilateral hemisphere also was determined on a posterior coronal section and compared with that of the contralateral hemisphere to ascertain the severity of cerebral atrophy/cavitation. Data were analyzed by linear models. CO2 tensions averaged 26, 42, 54, and 71 mm Hg in the 0, 3, 6, and 9% CO2 exposed animals, respectively, during systemic hypoxia. Blood O2 tensions during hypoxia were not different among the four groups and averaged 34.7 mm Hg. Neuropathologic results showed that 30/38 (79%) rats exposed to 3% CO2 showed either no or mild brain damage compared with 13/33 (39%) controls (0% CO2). Cystic cavitation occurred in only four CO2 exposed rat pups compared with 14 controls (P = .001). At 6% CO2 exposure, all of 20 rat pups showed either no damage or mild atrophy compared with controls (P < .001); and at 9% CO2 exposure, 19/23 (83%) rat pups showed no or mild damage compared with controls (P < .001). The data also showed that the greatest reduction in brain damage occurred in immature rats exposed to 6% CO2 with slightly less protection at 9% CO2 (P = .012), the latter comparable with the severity of brain damage sustained by animals inhaling 3% CO2. Analyses of coronal width ratios at each CO2 exposure provided results comparable with those of the gross neuropathology scores. The results indicate that in an immature rat model normocapnic cerebral hypoxia-ischemia is associated with less severe brain damage than in hypocapnic hypoxia-ischemia and that mild hypercapnia is more protective than normocapnia. The findings in an experimental model merit further animal investigations as well as a clinical reappraisal of the ventilatory management of sick newborn human infants.

Effect of hypoxia/ischemia on bicuculline-induced seizures in immature rats: behavioral and electrocortical phenomena.

The relation between hypoxia/ischemia and subsequent alterations in seizure susceptibility in developing brain remains unclear. We assessed the behavioral and electrocorticographic (ECoG) effects of hypoxic/ischemic brain damage on bicuculline (BIC)-induced seizures in 7-day postnatal rats, and determined maturational changes in seizure susceptibility, behavior and ECoG activity. Rat pups were subjected to unilateral common carotid artery ligation, followed by exposure to 8% O2 at 37 degrees C for 2 h, an insult that produces brain damage in the cerebral hemisphere ipsilateral to carotid artery occlusion. The experimental group consisted of rat pups previously subjected to hypoxia/ischemia; control littermates received neither arterial ligation nor systemic hypoxia. Experimental animals received 4, 5, or 6 mg/kg BIC subcutaneously (s.c.) at 2 and 24 h, and at 3, 7, and 21 days of recovery from hypoxia/ischemia. Two animals at each interval of recovery, 1 each from the experimental and control groups, were used for ECoG monitoring. After BIC injection, animal behavior was observed for 2 h. Behaviors and seizures were classified in five categories based on severity, duration, and character: 1, mild irritability; 2, few clonic seizures and agitation; 3, few tonic-clonic seizures with swimming movements; 4, frequent tonic-clonic seizures with apneic episodes; 5, continuous tonic-clonic seizures and death. Rat pups previously subjected to hypoxia/ischemia had lesser seizure susceptibility than controls at 2-h recovery (p < 0.05) and greater susceptibility than controls at 24 h (p < 0.05). Tonic seizures were prominent at 2 and 24 h in both the experimental and control groups, whereas lesion-sided circling was prominent only in the hypoxic/ischemic rat pups.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypoxic-ischemic injury in the neonatal rat brain: effects of pre-and post-treatment with the glutamate release inhibitor BW1003C87

In a model of perinatal hypoxia-ischemia (HI) we examined the neuroprotective efficacy of pre- and post-treatment with the glutamate release inhibitor BW1003C87 [5-(2,3,5-trichlorophenyl)-2,4-diamino-pyrimidine). Ipsilateral brain damage developed in 99% of rat pups subjected to HI (unilateral common carotid artery ligation and 100 min of 7.7% oxygen exposure) with a 26 ± 16% (mean ± S.D.) weight deficit of the damaged hemisphere 2 weeks after the insult. Pre-treatment with BW1003C87 (10 mg/kg intraperitoneally) reduced the brain damage by 46% ( P < 0.05). A higher dose (20 mg/kg) of pre-treatment was not tolerated. Administration of BW1003C87 did not affect the rectal temperature of the rats. Post-treatment with BW1003C87 (10–30 mg/kg) offered no neuroprotection in this model. In conclusion, there was a neuroprotective effect from pre- but not post-treatment with BW1003C87 in this model, supporting the concept that intra-ischemic excitatory amino acid release is important for development of brain damage. The lack of post-treatment effect indicates that BW1003C87 did not attenuate deleterious EAA cycling during reflow in the neonatal brain.

Cerebral Glucose and Energy Utilization during the Evolution of Hypoxic—Ischemic Brain Damage in the Immature Rat

The cerebral metabolic rate for glucose (CMRg1) and cerebral energy utilization (CEU) were assessed in immature rats during recovery from cerebral hypoxia-ischemia. CMRg1 was determined using a modification of the Sokoloff technique with 2-deoxy-[14C]glucose (2-DG) as the radioactive tracer. CEU was determined using the Lowry decapitation technique. Seven-day postnatal rats underwent unilateral common carotid artery ligation, followed 4 h thereafter by exposure to 8% oxygen at 37 degrees C for 3 h. At 1, 4, or 24 h of recovery, the rat pups underwent those procedures necessary for the measurement of either CMRg1 or CEU. At 1 h of recovery, the CMRg1 of the cerebral hemisphere ipsilateral to the carotid artery occlusion was 97% of the control rate (8.7 mumol 100 g-1 min-1) but was only 48% of the control in the contralateral hemisphere. At 4 h of recovery, the CMRg1 was increased 49% above baseline in the ipsilateral hemisphere, decreasing thereafter to 84% of the control at 24 h. The CMRg1 of the contralateral hemisphere normalized by 4 h of recovery. An inverse correlation between endogenous concentrations of ATP or phosphocreatine and CMRg1 in the ipsilateral hemisphere was apparent at 4 h of recovery. CEU in the ipsilateral cerebral hemisphere was 64 and 46% of the control (3.47 mmol approximately P/kg/min) at 1 and 24 h, respectively (p < 0.05) and 77% of the control at 4 h of recovery. CEU in the contralateral hemisphere was unchanged from the control at all measured intervals. Correlation of the alterations in CMRg1 with those in CEU at the same intervals indicated that substrate supply exceeds energy utilization during early recovery from hypoxia-ischemia. The discrepancy combined with a persistent disruption of the cerebral energy state implies the existence of an uncoupling of mitochondrial oxidative phosphorylation as one mechanism for the occurrence of perinatal hypoxic-ischemic brain damage.

Effect of unilateral perinatal hypoxic-ischemic brain damage on the gross development of opposite cerebral hemisphere.

The effect of unilateral perinatal cerebral hypoxic-ischemic damage on the gross development of the opposite hemisphere was investigated in immature rats. Eight-day-old rats underwent unilateral common carotid artery ligation followed by exposure to hypoxia (8% oxygen) ranging from 1 to 2 h at 37 degrees C. The animals were sacrificed 3 weeks later, and brain damage was assessed histologically, by weighing the cerebral hemispheres, and by measuring hemispheric cross-sectional area and diameter as well as the thickness of neocortex. There were no significant differences in either the size or cortical thickness of the cerebral hemisphere contralateral to the damaged hemisphere compared to those of controls. The results are comparable to the effect of unilateral perinatal hemispheric decortication and hemispherectomy on the growth of the contralateral hemisphere and suggest that: (1) perinatal damage to one cerebral hemisphere does not significantly alter the size of the other hemisphere, and (2) the contralateral hemisphere may be used as a 'normal' reference in evaluating the extent of atrophy or infarction of a cerebral hemisphere damaged earlier by perinatal hypoxia-ischemia.

Influence of mild hypothermia on hypoxic-ischemic brain damage in the immature rat.

Recent studies in adult animals have shown that even small decreases in brain or core temperature ameliorate the damage resulting from hypoxic-ischemic insults. To determine the effect of minor reductions in ambient temperature either during or after an hypoxic-ischemic insult on the brain of the immature rat, 7-d-postnatal rat pups underwent unilateral common carotid artery ligation followed by exposure to hypoxia in 8% oxygen for 3 h. Control animals were maintained at 37 degrees C during hypoxia-ischemia. Intraischemic hypothermia was induced during the insult at temperatures of 34 degrees C and 31 degrees C. Postischemic hypothermia was induced by exposing rat pups that underwent hypoxia at 37 degrees C to recovering environments of 34 degrees C and 31 degrees C. Temperatures were recorded every 15 min from thermistor probes placed in the ipsilateral hemisphere and rectally. Neuropathologic alterations were assessed at 30 postnatal d. During hypoxia, animals became poikilothermic. Brain damage occurred in 90% of rat pups exposed to hypoxia-ischemia at 37 degrees C. Cerebral injury significantly decreased with decreasing temperatures during hypoxia-ischemia (p < 0.01). Only 30% of rats had brain damage when exposed to hypoxia-ischemia at 34 degrees C, and none of the rats exposed at 31 degrees C had brain damage. In contrast, there was no difference in the extent of cerebral injury between rat pups recovered under hypothermic conditions of either 34 degrees C or 31 degrees C compared with those recovered at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Immediate early gene induction after neonatal hypoxia-ischemia

Immediate early gene (IEG) products, such as FOS and JUN, may partially mediate the long-term transcriptional response of CNS cells to specific changes in their environment. To determine whether IEG products might be involved in the immature brain's response to hypoxia-ischemia (H-I), 7-day-old rat pups were subjected to unilateral common carotid artery ligation followed by 3 h of hypoxia (8% O 2/92% N 2) at 37°C, which results in pathological changes only in specific regions of the hemisphere ipsilateral to ligation 49. Time course experiments were performed, in which animals were sacrificed between 1 and 24 h after H-I. RNAs from several brain regions were analyzed by Northern blot hybridization for their relative concentrations of nine IEG mRNAs (c-fos, c-jun, junB, TIS 1 (nur77), TIS7, TIS8 (zif268), TIS10, TIS11, and TIS21). Induction of all IEGs, except TIS7 and TIS10, was observed in ipsilateral forebrain, and, less frequently, in contralateral forebrain, at 1, 2, and 3 h post-hypoxia. In some animals, lower levels of expression were also detected at 4, 18 and 24 h. With minor exceptions, co-induction of all seven IEGs was observed in a given RNA sample. Induction of two other mRNAs, representing the heat shock and astrocytic responses, were also observed. Hsp70 mRNA levels were increased only in the brains of animals exhibiting IEG induction. However, hsp70 induction was confined to the ipsilateral forebrain, implying a more direct relationship between its expression and permanent morphological damage. GFAP mRNA induction occurred predominantly in ipsilateral forebrain samples at 18 and 24 h post-hypoxia. Levels of B-actin and ubiquitin mRNAs were relatively constant in the same RNA samples. In control experiments c-fos mRNA induction was not detected after sham ligation with hypoxia, ligation with sham hypoxia, or hypoxia alone. These results suggest that the immature brain is highly responsive to H-I at the level of gene expression, involving at least three different rapid response systems.

The Level of Thrombus Formation in Experimental Carotid Artery Occlusion Done by Ligation and Endarterectomy Method

The purpose of this study was to develop thrombus formation by ligation and endarterectomy (L + E) in carotid artery in rats. Unilateral common carotid artery (CCA) ligation and endarterectomy were performed in 15 rats with microsurgical techniques, and 15 rats were used as a control group. The thrombus formation was examined after days 1, 3, and 7 and months 1 and 3. Local thrombus was revealed in ligation of the common carotid artery. The thrombus formation did not extend further than the bifurcation of the CCA in the L + E group.

Cerebral energy metabolism during hypoxia-ischemia and early recovery in immature rats.

Persistent alterations in cellular energy homeostasis may contribute to the brain damage that evolves from perinatal cerebral hypoxia-ischemia. Accordingly, the presence and extent of perturbations in high-energy phosphate reserves were analyzed during hypoxia-ischemia and the early recovery period in the immature rat. Seven-day postnatal rats were subjected to unilateral common carotid artery ligation and hypoxia with 8% oxygen at 37 degrees C for 3 h, an insult that produces damage (selective neuronal necrosis or infarction) of the cerebral hemisphere ipsilateral to the common carotid artery ligation in 92% of animals. Rat pups were quick frozen in liquid nitrogen during hypoxia-ischemia and at 10, 30, and 60 min and 4 and 24 h of recovery for enzymatic, fluorometric analysis of phosphocreatine (PCr), creatine, ATP, ADP, and AMP. During hypoxia-ischemia, PCr, ATP, and total adenine nucleotides were decreased by 87, 72, and 50% of control, respectively. During recovery, PCr, ATP, and total adenine nucleotides exhibited a rapid (within 10 min) although incomplete and heterogeneous recovery that persisted for at least 24 h. Mean values for PCr remained between 55 and 85% of control, whereas ATP values remained between 57 and 67% of control. Individual ATP values were inversely related to tissue water content at 10 min of recovery, indicating a close correlation between failure of energy restoration and the extent of cerebral edema as a reflection of brain damage. Thus high-energy phosphate reserves display lingering alterations during recovery from hypoxia-ischemia. The interanimal variability in energy restoration presumably reflects the spectrum of brain damage seen in this model of perinatal cerebral hypoxia-ischemia.

Effect of insulin-induced and fasting hypoglycemia on perinatal hypoxic-ischemic brain damage.

Experiments in adult animals have indicated that hyperglycemia accentuates whereas hypoglycemia ameliorates hypoxic-ischemic brain damage. To determine whether hypoglycemia is protective or deleterious to the perinatal brain subjected to hypoxia-ischemia, 7-d postnatal rats were rendered hypoglycemic either by receiving an s.c. injection of insulin or fasting for 12 h. All rat pups underwent unilateral common carotid artery ligation followed by exposure to 8% oxygen-balance nitrogen at 37 degrees C for 2 h. Control animals (no insulin or fasting) received s.c. injections of normal saline. Mean blood glucose concentrations were 5.4 +/- 0.1, 4.3 +/- 0.2, and 3.4 +/- 0.1 mmol/L for control, insulin, and fasted animals, respectively. Blood beta-hydroxybutyrate concentrations were identical (0.5 +/- 0.1 mmol/L) for control and insulin-treated animals, but more than doubled in concentration in the fasted animals (p less than 0.001). Mortality rates during hypoxia-ischemia were higher in the insulin-treated animals (30%) than in either the fasted (4%) or control (0%) animals (p less than 0.05). Fasted animals showed a significant reduction in hypoxic-ischemic brain damage as compared with either the insulin-treated or control animals. Insulin-treated animals were not significantly different from controls. The findings indicate that 1) insulin induced hypoglycemia does not provide a protective effect on perinatal hypoxic-ischemic brain damage, as in adults; and 2) fasting adequate to produce hypoglycemia and ketonemia improved neuropathologic outcome.

Cerebral oxidative metabolism and redox state during hypoxia-ischemia and early recovery in immature rats

Intracellular pH (pHi) and cytoplasmic and mitochondrial oxidation-reduction (redox) states of cerebral tissue were examined in relation to perturbations of glycolytic and tricarboxylic acid cycle intermediates and of high-energy phosphate reserves during hypoxia-ischemia and the early recovery period in the immature rat. Seven-day postnatal rats underwent unilateral common carotid artery ligation and exposure to 8% O2 for 3 h, after which they were quick frozen in liquid N2 at the terminus of hypoxia-ischemia and at 10, 30, 60, and 240 min of recovery for enzymatic fluorometric analysis of cerebral metabolites. During hypoxia-ischemia, concentrations of glucose and alpha-ketoglutarate in the cerebral hemisphere ipsilateral to the carotid artery occlusion were depleted to 10 and 70% of control, respectively; pyruvate was unchanged. During recovery, glucose, pyruvate, and alpha-ketoglutarate increased above their respective control values. Calculated pHi decreased from 7.0 (control) to 6.6 during hypoxia-ischemia and normalized by 10 min of recovery. The cytoplasmic NAD+/NADH ratio decreased (increased reduction) to 50% of control during hypoxia-ischemia and remained in the reduced state throughout 4 h of recovery. Paradoxically, mitochondrial NAD+/NADH was oxidized at the terminus of hypoxia-ischemia. The mitochondrial oxidation which developed during hypoxia-ischemia presumably results from a limitation of cellular substrate (glucose) supply, which in turn leads to a depletion of high-energy phosphate reserves, culminating in brain damage.

Regional cerebral blood flow during hypoxia-ischemia in the immature rat: comparison of iodoantipyrine and iodoamphetamine as radioactive tracers

The indicator fractionation method was used to measure regional cerebral blood flow (rCBF) in immature rats, using either iodo-[ 14C]antipyrine (IAP) or isopropyl-[ 14C]iodoamphetamine (IPIA) as the radioisotope. Seven-day postnatal rats received a subcutaneous injection of either IAP (5 μCi) of IPIA (10 μCi), two minutes following which the animals were sacrificed and their brains prepared for quantitative autoradiography. Blood flows to cerebral cortex, hippocampus, striatum, thalamus and hypothalamus were comparable using the two methods, whereas blood flows to white matter structures were substantially higher (+ 64–85%) with IAP. Spatial resolution, especially of the gray matter structures, was greater with IPIA than with IAP. During cerebral hypoxia-ischemia (unilateral common carotid artery ligation and hypoxia with 8% oxygen), blood flows to all regions of the ipsilateral cerebral hemisphere were not different from control at 10 and 20 min. At 1–3 h of hypoxia-ischemia, blood flows were decreased in all analyzed structures of the cerebral hemisphere ipsilateral to the carotid artery occlusion, ranging from 7% to 40% of control values. A columnar or patchy distribution of preferential perfusion was seen within the cerebral cortex, striatum and thalamus, with IPIA, which corresponds closely to the pathologic pattern of injury seen within these structures of the immature rat. Such a preferential perfusion was not seen in hippocampus, in which metabolic factors (intrinsic vulnerability) most likely predict the distribution of hypoxic-ischemic brain damage.