Unilateral Common Carotid Artery Ligation Research Articles

Tissue factor pathway inhibitor deficiency enhances neointimal proliferation and formation in a murine model of vascular remodelling

Tissue factor (TF) is a small-molecular-weight glycoprotein that initiates the extrinsic coagulation pathway but may have important noncoagulation vascular functions as well. Tissue factor pathway inhibitor (TFPI) is a major physiological inhibitor of TF-initiated coagulation. Enhancement of vascular TFPI either by overexpression using gene transfer or delivery of protein to the vessel has been shown to reduce neointimal formation. However, the inherent role of TFPI in this process has not been defined. To do so, we utilized a murine model of vascular remodeling using flow cessation in mice, which are heterozygous for a genetic deletion of the first Kunitz domain of TFPI or wild type littermates. The heterozygotic mice had 50% of wild type TFPI activity in plasma as well as vascular homogenates. To study the effect of TFPI deficiency on neointimal formation, age matched TFPI(K1)+/- and wildtype littermates underwent unilateral common carotid artery ligation. Mice were sacrificed at 4 weeks and the ligated carotid arteries were analyzed. There was a significantly greater neointima to media ratio and less luminal area in the TFPI(K1)+/- mice compared to their TFPI(K1)+/+ littermates. The proliferative index of intimal cells in TFPI(K1)+/- mice at 1 week was significantly higher compared to TFPI(K1)+/+ mice. We conclude that TFPI deficiency enhances neointimal formation and proliferation associated with flow cessation. This suggests that TFPI may regulate vascular remodeling primarily through modulation of neointimal formation.

Interleukin-10 Fails to Modulate Low Shear Stress–Induced Neointimal Hyperplasia

Introduction. Overexpression of the anti-inflammatory cytokine interleukin-10 (IL-10) blocks atherosclerotic events in vivo, and IL-10 has been recently hailed as an “immunologic scalpel” for atherosclerosis. Alternatively, mice lacking IL-10 receiving atherogenic diets have increased occlusive lesions. It remains unclear whether such IL-10 modulation broadly applies to other forms of occlusive arterial remodeling. We hypothesized that lack of IL-10 would exacerbate, and exogenous or overexpression of IL-10 would abrogate low shear stress–induced neointimal hyperplasia (NIH).Methods. Wild-type (WT) and IL-10-deficient (IL-10−/−) mice underwent unilateral common carotid artery (CCA) ligation. Low shear stress in the patent ligated artery results in remodeling and formation of neointima containing BrdU and SMC α-actin-positive cells. Additional groups of WT mice underwent CCA ligation and were treated daily with intraperitoneal saline or 1 μg of human IL-10. Chronic delivery gene therapy approaches were also utilized to define the role of IL-10 signaling. WT mice were treated adventitially with 1 × 1010 adenovirus/green fluorescent protein (Ad/gfp) and an Ad/empty control to confirm the veracity of adventitial delivery. Then, Ad viral IL-10 (vIL-10), Ad/empty, and virus buffer alone were applied directly to the adventitia of the CAA immediately following ligation. In separate experiments, 1 × 1010 Ad/empty or Ad/vIL-10 was injected intramuscularly. CCAs were perfusion fixed 28 days postligation, the time at which NIH is near maximum.Results. IL-10−/− mice developed identical NIH areas compared to WT controls. Mice receiving IL-10 demonstrated NIH equivalent to saline controls. Mice receiving intramuscular or adventitial Ad/IL-10 developed high serum levels of IL-10 yet formed NIH areas similar to those of controls. Serum IL-10 levels were significantly higher (P = 0.04) with adventitial delivery. Mice treated adventitially with Ad/gfp showed reliable transfection of cells within the adventitia of CAA. No antibody to human IL-10 was found in the sera of intraperitoneal IL-10-treated mice, which failed to attenuate NIH.Conclusion. Under the conditions of this experiment, lack of IL-10 does not exacerbate low shear stress–induced NIH, nor does exogenous administration or overexpression of IL-10 attenuate it. Despite high serum levels of vIL-10 in mice treated with ad/vIL-10 adventitially, there appears to be no therapeutic effect despite the confirmed transfection of adventitial cells. Discrepancies between these findings and previous research may be related to IL-10 dosing, inflammation induced by the adenoviral vector, or disparities between the NIH models.

Role of excitatory amino acid transporter 1 in neonatal rat neuronal damage induced by hypoxia-ischemia

The role of excitatory amino acid transporter 1 in neonatal rat neuronal damage was studied following hypoxia-ischemia. To induce hypoxia-ischemia injury, rats on postnatal day 7 were exposed to 8% oxygen for 2 h following unilateral common carotid artery ligation. According to brain damage scoring based on Cresyl Violet staining, the neuronal damage time-dependently changed in the ischemic regions following hypoxia-ischemia. Immunohistochemical studies showed that excitatory amino acid transporter 1 expression was mainly observed in the cerebral cortex ipsilateral to common carotid artery ligation and markedly increased at 24 h and 48 h following hypoxia-ischemia. Combined with confocal laser scanning microscopic analysis, double staining showed that excitatory amino acid transporter 1 positive staining appeared in neurons as well as astrocytes after hypoxia-ischemia. Most excitatory amino acid transporter 1 positive staining cells exhibited regular morphological characteristics and only a few were double-stained by terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick-end labeling. Down-regulation of excitatory amino acid transporter 1 expression by intraventricular administration of specific antisense oligonucleotide exacerbated neuronal damage in hypoxia-ischemia brain. These results suggest that the increase of excitatory amino acid transporter 1 expression may be involved in a pathophysiological process of hypoxia-ischemia brain damage and may reflect a self-compensative mechanism for protecting neurons from further injury.

Intracellular calcium accumulation during the evolution of hypoxic–ischemic brain damage in the immature rat

An excessive intracellular accumulation of calcium (Ca 2+) in neurons and glia has been proposed to represent a major ‘final common pathway’ for cell death arising from hypoxia–ischemia. To clarify the role of altered calcium flux into the perinatal brain undergoing hypoxic–ischemic damage, 7-day postnatal rats underwent unilateral common carotid artery ligation followed by systemic hypoxia with 8% oxygen. This insult is known to produce brain damage in the form of selective neuronal death or infarction largely limited to the cerebral hemisphere ipsilateral to the arterial occlusion. Either prior to or following hypoxia–ischemia, the rat pups received a s.c. injection of 45CaCl 2, and specimens of blood, cerebrospinal fluid (CSF), and brain were obtained for isotopic measurements and the calculation of the extent of brain intracellular radioactivity. During hypoxia–ischemia, there was a modest increase in intracellular Ca 2+ radioactivity (+28–47%) in both cerebral hemispheres only after 2 h of hypoxia–ischemia. During recovery from 2 h of hypoxia–ischemia, intracellular Ca 2+ accumulated progressively only in the ipsilateral cerebral hemisphere for up to 24 h, during which interval intracellular Ca 2+ decreased in the contralateral hemisphere. No such progressive accumulation was noted during recovery in animals previously exposed to only 1 h of hypoxia–ischemia. The results suggest that a disruption of intracellular Ca 2+ homeostasis is a major contributing factor in the evolution of perinatal hypoxic–ischemic brain damage. Ca 2+ accumulation is a relatively modest and late event during the hypoxic–ischemic phase, and a progressive overload occurs during the recovery phase only if infarction occurs. The question remains as to whether or not the intracellular Ca 2+ overload occurring during recovery is a contributor to or a consequence of the ultimate brain damage.

Experimental stroke in the female diabetic, db/db, mouse.

Diabetic hyperglycemia increases brain damage after cerebral ischemia in animals and humans, although the underlying mechanisms remain unclear. Gender-linked differences in ischemic tolerance have been described but have not been studied in the context of diabetes. In the current study, we used a model of unilateral common carotid artery ligation, combined with systemic hypoxia, to study the effects of diabetes and gender on hypoxic-ischemic (HI) brain damage in the genetic model of Type II diabetes, the db/db, mouse. Male and female, control and db/db, mice were subjected to right common carotid artery ligation followed by varying periods of hypoxia (8% oxygen/92% nitrogen) to assess mortality, infarct volume, and tissue damage by light microscopic techniques. End-ischemic regional cerebral blood flow (CBF) was determined using [14C] iodoantipyrine autoradiography. Glycolytic and high energy phosphate compounds were measured in blood and brain by enzymatic and fluorometric techniques. Gender and diabetes had significant effects on mortality from HI and extent of brain damage in the survivors. Female mice were more resistant than their male counterparts, such that the severity (mortality and infarction size) in the male diabetics > female diabetics - male controls > female controls. Endischemic CBF and depletion of cerebral high energy reserves were comparable among all groups. Surprisingly, female diabetic mice were more hyperglycemic and demonstrated a greater prolonged lactacidosis than the males; however, they were more resistant to damage. The results suggest a unique pathophysiology of hypoxia-ischemia in the female diabetic brain.

Direct evidence for cytokine involvement in neointimal hyperplasia.

Tumor necrosis factor-alpha (TNF-alpha) and interleukin 1 (IL-1) are proximal inflammatory cytokines that stimulate expression of adhesion molecules and induce synthesis of other proinflammatory cytokines. In addition, TNF-alpha and IL-1 influence vascular smooth muscle cell migration and proliferation in vitro. In view of the inflammatory nature of neointimal hyperplasia (NIH), we tested the hypothesis that endogenous TNF-alpha and IL-1 modulate low shear stress-induced NIH. Mice underwent unilateral common carotid artery (CCA) ligation. Low shear stress in the patent ligated CCA has previously been shown to result in remodeling and NIH. Reverse transcriptase-polymerase chain reaction for TNF-alpha and IL-1alpha mRNA demonstrated both TNF-alpha and IL-1alpha mRNA in ligated CCAs, whereas normal and sham-operated CCAs had none. Mice lacking functional TNF-alpha (TNF-/-) developed 14-fold less neointimal area than WT controls (P:<0.05). p80 IL-1 type I receptor knockout (IL-1RI-/-) mice tended to develop less (7-fold, P:>0.05) neointimal area than WT controls. Furthermore, no IL-1alpha mRNA expression was detected in CCAs from TNF-/- mice; however, TNF-alpha mRNA expression was found in the IL-1RI-/- mice. Mice that overexpress membrane-bound TNF-alpha but produce no soluble TNF-alpha display an accentuated fibroproliferative response to low shear stress (P:<0.05). These results directly demonstrate that TNF-alpha and IL-1 modulate NIH induced by low shear stress. NIH can proceed by way of soluble TNF-alpha-independent mechanisms. Specific anti-TNF-alpha and anti-IL-1 therapies may lessen NIH.

High-dose S(+)-ketamine improves neurological outcome following incomplete cerebral ischemia in rats.

To determine the effects of the non-competitive NMDA-receptor antagonist S(+)-ketamine on neurological outcome in a rat model of incomplete cerebral ischemia. Thirty rats were anesthetized, intubated and mechanically ventilated with isoflurane, O2 30% and nitrous oxide 70%. Following surgery animals were randomly assigned to one of the following treatment groups: Rats in group 1 (n = 10,OFF control) received fentanyl (bolus: 10 microg x kg(-1) i.v.; infusion 25 microg x kg(-1) x h(-1)) and N2O 70% / O2. Rats in group 2 (n = 10) received O2 30% in air and low-dose S(+)-ketamine (infusion: 0.25 mg x kg(-1) x min(-1)). Rats in group 3 (n = 10) received O2 30% in air and high-dose S(+)-ketamine (infusion: 1.0 mg x kg(-1) min(-1)). Following 30 min equilibration period ischemia was induced by combined unilateral common carotid artery ligation and hemorrhagic hypotension to 35 mm Hg for 30 min. Plasma catecholamines were assayed before and at the end of ischemia. Neurological deficit was evaluated for three postischemic days. Neurological outcome was improved with high-dose S(+)-ketamine when compared to fentanyl / N2O -anesthetized controls (9 vs. 1 stroke related deaths, P<0.05). Increases in plasma catecholamine concentrations were higher in fentanyl / N2O -anesthetized (adrenaline baseline 105.5+/-92.1 pg x ml(-1), during ischemia 948+/-602.8 pg x ml(-1), P<0.05; noradrenaline baseline 407+/-120.2 pg x ml(-1), ischemia 1267+/-422.2 pg x ml(-1), P <0.05) than in high-dose S(+)-ketamine-treated animals (adrenaline baseline 71+/-79.5 pg x ml(-1), ischemia 237 +/-131.9; noradrenaline baseline 317.9+/-310.5 pg x ml(-1), ischemia 310.5+/-85.7 pg x ml(-1)). Neurological outcome is improved following incomplete cerebral ischemia with S(+)-ketamine. Decreases in neuronal injury may be related to suppression of sympathetic discharge.

Reduced nitric oxide is involved in prenatal ischemia-induced tolerance to neonatal hypoxic-ischemic brain injury in rats

To explore the role of nitric oxide (NO) in the hypoxic-ischemic (HI) tolerance phenomenon, NO production and brain injury following neonatal hypoxia-ischemia (induced by unilateral common carotid artery ligation followed by hypoxic exposure) were assessed in rat pups with or without HI preconditioning. A previously demonstrated prenatal HI rat model of preconditioning was used in this study. On G17, rat fetuses were subjected to either HI in utero (PreHI) for 30 min or a sham operation (SH). The PreHI treatment provided significant protection against neonatal HI-induced brain injury, as indicated by decreased ipsilateral brain weight reduction, less severe tissue damage, and decreased activation of caspase-3. Concomitant with the protective effect of prenatal HI preconditioning, elevation of nitrite/nitrate content in the ipsilateral cortex of the brain, as an indirect measure of NO production, was significantly lower in the PreHI group than in the SH group following neonatal HI. The protective effect of prenatal HI preconditioning could be reversed by sodium nitroprusside (SNP), a spontaneous NO donor, while SNP had no effect on neonatal HI-induced brain injury in the SH group. Intraperitoneal administration of SNP to pups from the PreHI group (2 mg/kg, 24 and 1.5 h before neonatal HI) increased neonatal HI-induced brain injury similar to that observed in the SH group. On the other hand, l-N G-nitro-arginine (2 mg/kg, i.p., 1.5 h before the hypoxic exposure), an NO synthase inhibitor, significantly attenuated neonatal HI-induced brain injury in the SH group. The overall results indicate that reduced NO production in the preconditioned rat brain contributes to prenatal HI-induced tolerance to neonatal HI brain injury.

Protection of neonatal rat brain from hypoxic-ischemic injury by LY379268, a Group II metabotropic glutamate receptor agonist.

Neuroprotective effects of a Group II metabotropic glutamate receptor agonist, LY379268, were examined in a neonatal rat model of hypoxia-ischemia (unilateral common carotid artery ligation followed by hypoxic exposure for 1.5h in 7-day-old rat pups). LY379268 administered 5 min after hypoxic exposure (2, 5, or 10 mg/kg, i.p.) significantly reduced brain injury as measured by reductions in the ipsilateral brain weight and in CA1 hippocampal neuron density. The significant neuroprotective effects were also observed when this compound (5 mg/kg) was administered 30 min, but not 60 min, after hypoxic exposure. The neonatal hypoxia-ischemia (HI) procedure significantly increased caspase-3 activity and induced DNA fragmentation in the ipsilateral cortex compared with that in the contralateral cortex 24 and 72h after the insult, respectively. LY379268 did not prevent this increase in caspase-3 activity and DNA fragmentation in the ipsilateral cortex. These results suggest that activation of Group II metabotropic glutamate receptors may provide neuroprotection against HI brain injury. However, blockade of caspase-3 activation and the apoptotic pathway appears not to be involved in the neuroprotective effects of LY379268 observed in the neonatal rat model of HI.

CSF glutamate during hypoxia–ischemia in the immature rat

Cerebrospinal fluid (CSF) glutamate was measured prior to and during the course of cerebral hypoxia–ischemia in the immature rat to estimate its concentration in the extracellular fluid (ECF). A preliminary experiment was conducted using [ 14 C ]glutamate injections into immature rat brain, which showed that equilibration between ECF and CSF occurred within 10 min. Seven-day postnatal rats underwent unilateral common carotid artery ligation followed by hypoxia with 8% oxygen for up to 2 h. Brain damage, in the form of selective neuronal necrosis or apoptosis, commences after 60 min, while infarction commences after 90 min of hypoxia–ischemia. During the course of hypoxia–ischemia, CSF was obtained from the cisterna magna and analyzed for glutamate. No statistically significant increases in CSF glutamate occurred until 105 min, at which time the concentration was 240% of control (20 μmol/l). By 120 min, CSF glutamate had increased over twofold above the control value. In rat pups exposed to 1 h of hypoxia–ischemia, no increases in CSF glutamate occurred for up to 6 h of recovery. In animals exposed to 2 h of hypoxia–ischemia, CSF glutamate decreased to the control value by 1 h of recovery, with a secondary rise at 6 h. Accordingly, the increase in CSF, and presumably ECF, glutamate is a late event, which better corresponds temporally to cerebral infarction than to selective neuronal death. The results suggest that glutamate excitotoxicity, although involved in the occurrence of infarction, neither causes or contributes to selected neuronal death. The secondary elevation in CSF glutamate at 6 h of recovery from 2 h of hypoxia–ischemia occurs coincident with the onset of tissue necrosis, seen histologically.

Serial magnetic resonance diffusion and hemodynamic imaging in a neonatal rabbit model of hypoxic-ischemic encephalopathy.

Dynamic changes in relative cerebral blood volume (rCBV) and apparent diffusion coefficient (ADC) were investigated, using high speed magnetic resonance imaging (MRI) in an acute neonatal rabbit model of hypoxic-ischemic encephalopathy (HIE). Serial rCBV imaging used a magnetic susceptibility blood pool contrast agent. Interleaved ADC and rCBV images were acquired with 9 s temporal resolution. Rabbits received unilateral common carotid artery (CCA) ligation followed by hypoxia. rCBV increased bilaterally within 1-2 min after the onset of hypoxia. A biphasic ADC decline was observed: a slowly declining phase (84 +/- 18% of baseline) followed by a rapid, focal drop to 55 +/- 8% of baseline in the ipsilateral cortex, which was paralleled by a rapid focal rCBV drop to 70 +/- 17% of baseline. ADC decline generally began in a small region of ipsilateral cortex and spread over the ipsilateral cortex, ipsilateral subcortical tissue and contralateral cortex. The initial ADC drop usually preceded the initial rCBV drop by approximately 60 s, however at later timepoints rCBV decline sometimes preceded ADC decline. Upon normoxia, rCBV recovered to about baseline values while ADC recovered to baseline or above. This method provides a sensitive means of non-invasively visualizing acute hemodynamic- and metabolic-related changes in HIE with good temporal and spatial resolution.

Desflurane and isoflurane improve neurological outcome after incomplete cerebral ischaemia in rats

We have investigated the effects of isoflurane and desflurane on neurological outcome in a rat model of incomplete cerebral ischaemia. We studied 40 non-fasted male Sprague-Dawley rats, anaesthetized, intubated and ventilated mechanically with isoflurane and nitrous oxide in oxygen (FlO2 0.3). Arterial and venous catheters were inserted for measurement of arterial pressure, drug administration and blood sampling. A biparietal electroencephalogram (EEG) was recorded continuously using subdermal platinum electrodes. At completion of surgery, administration of isoflurane was discontinued (with the exception of those animals receiving isoflurane as treatment) and rats were allowed an equilibration period of 30 min according to the following procedure: group 1 (n = 10), 66% nitrous oxide in oxygen and fentanyl (bolus 10 micrograms kg-1 i.v. followed by infusion at a rate of 25 micrograms kg-1 h-1); group 2 (n = 10), 1.0 MAC of isoflurane in oxygen (FlO2 0.3) and air; groups 3 and 4 (n = 10 per group), 1.0 MAC or 1.5 MAC of desflurane in oxygen (FlO2 0.3) and air, respectively. Ischaemia was produced by combined unilateral common carotid artery ligation and haemorrhagic hypotension to 35 mm Hg for 30 min. Functional neurological deficit was evaluated for 3 days after cerebral ischaemia. At baseline, brain electrical activity was higher with fentanyl-nitrous oxide, 1.0 MAC of isoflurane and 1.0 MAC of desflurane (groups 1-3) compared with 1.5 MAC of desflurane (group 4). Neurological outcome was improved in isoflurane and desflurane anaesthetized animals (groups 2-4), regardless of the concentration used compared with fentanyl-nitrous oxide anaesthesia (group 1). The increase in plasma epinephrine and norepinephrine concentrations during ischaemia was significantly higher in fentanyl-nitrous oxide anaesthetized animals (group 1) compared with animals who received volatile anaesthetics (groups 2-4). These data suggest that cerebral protection produced by isoflurane and desflurane appears to be related to reduction in sympathetic activity rather than suppression of cerebral metabolic rate.

Heat shock protein 72 expression and microtubule-associated protein 2 disappearance after hypoxia-ischemia in the developing rat brain

Objective: This study was intended to investigate the temporal changes in heat shock protein 72 expression and microtubule-associated protein 2 disappearance in rat brain at 2 different ages after hypoxic-ischemic insult. Study Design: Both 5-day-old and 14-day-old Wistar rats were subjected to unilateral common carotid artery ligation and hypoxia in 8% oxygen for 2 hours at 33°C. Brain sections were examined sequentially for heat shock protein 72 expression at 0.5, 3, 6, 12, 24, 48, and 72 hours of recovery after hypoxia-ischemia and for microtubule-associated protein 2 disappearance at 0, 24, 48, and 72 hours of recovery and at 7 days of recovery after hypoxia-ischemia. Results of immunohistochemical staining for heat shock protein 72 and microtubule-associated protein 2 were used as markers for detection of early hypoxic-ischemic brain damage. Permanent neuronal damage was assessed with hematoxylin and eosin staining at 7 days after hypoxia. Results: In 5-day-old rats microtubule-associated protein 2 expression was lost as early as 0 hours after hypoxia-ischemia in the cerebral cortex and hippocampus, with a peak at 48 hours after which expression recovered. Expression of heat shock protein 72 was detected in the ligated hemisphere at 0.5 hours after hypoxia-ischemia and peaked at 6 to 24 hours of recovery. In 14-day-old rats microtubule-associated protein 2 was stained in the cortex at 0 hours after hypoxia-ischemia but gradually disappeared in the cerebral cortex and hippocampus after 24 hours of recovery. The expression of heat shock protein 72 was not detected by 6 hours of recovery in the cerebral cortex and by 3 to 12 hours of recovery in the hippocampus, but heat shock protein 72 was persistently expressed in the cortex and hippocampus after 48 hours of recovery. Neuronal damage was significantly less in 5-day-old rats than in 14-day-old rats. Conclusion: In 5-day-old rats hypoxia-ischemia causes earlier changes in heat shock protein 72 and microtubule-associated protein 2 immunostaining results and causes less severe brain damage than in 14-day-old rats. (Am J Obstet Gynecol 1999;180:1254-62.)

Effect of seizures on cerebral hypoxic–ischemic lesions in immature rats

The present investigation was designed to study the effect of chemically induced seizures on cerebral hypoxic–ischemic (HI) damage in immature animals. Accordingly, cerebral HI was produced in 7-day postnatal (p7) rats and p13 rats by combined unilateral common carotid artery ligation and hypoxia with 8% oxygen. Seizures were induced chemically by the subcutaneous injection of kainic acid (KA) or inhalation of flurothyl vapor. Three types of experiments were conducted in each age group and for each convulsant. In some animals (group 1), seizures were produced at 24 h and again at 6 h prior to HI. In groups 2 and 3, seizures were induced 2 h or 24 h post HI, respectively. The results indicate that in group 1 animals, the first seizure significantly reduced duration of the second seizure challenge 18 h later at both p7 and p13 ( p=0.001). Histologic examination of brains of animals in group 1 subjected to seizures prior to HI and their HI-only controls showed that seizures prior to HI conferred protection against cerebral damage. This effect was significant for flurothyl seizures in p13 rats for all cerebral regions, especially hippocampal CA1 ( p=0.0004), and in p7 rats for hippocampus ( p=0.04) and particularly cerebral cortex ( p=0.007). For KA seizures, the protective effect was only significant in p13 rats and was limited to hippocampal CA regions and subiculum ( p=0.0009). Histologic assessment of cerebral lesions of p7 and p13 rats in the other two groups showed no significant difference between the animals subjected to seizures 2 h or 24 h post HI and their HI-only controls ( p>0.05). In conclusion, the results of the present study provide no evidence that seizures in early postnatal development aggravate pre-existing cerebral HI damage. They do suggest that seizures prior to HI or prior to a second seizure confer tolerance to both conditions.

Hypoxic preconditioning and hypoxic-ischemic brain damage in the immature rat: pathologic and metabolic correlates.

It has been reported that immature rats subjected to cerebral hypoxia-ischemia sustain less brain damage if they are previously exposed to systemic hypoxia compared with animals not exposed to prior hypoxia. Accordingly, neuropathologic and metabolic experiments were conducted to confirm and extend the observation that hypoxic preconditioning protects the perinatal brain from subsequent hypoxic-ischemic brain damage. Six-day postnatal rats were subjected to systemic hypoxia with 8% oxygen at 37 degrees C for 2.5 h. Twenty-four hours later, they were exposed to unilateral cerebral hypoxia-ischemia for 2.5 h, produced by unilateral common carotid artery ligation and systemic hypoxia with 8% oxygen. Neuropathologic analysis, conducted at 30 days of postnatal age, indicated a substantial reduction in the severity of brain damage in the preconditioned rats, such that only 6 of 14 such animals exhibited cystic infarction, but all 13 animals without prior preconditioning exhibited infarction (p < 0.001). Measurement of cerebral glycolytic and tricarboxylic acid intermediates and high-energy phosphate reserves at the terminus of and at 4 and 24 h following hypoxia-ischemia showed no differences in the extent of alterations in the preconditioned and nonpreconditioned immature rats. A difference was seen in the restitution of high-energy stores during the first 24 h of recovery from hypoxia-ischemia, with a more optimal preservation of these metabolites in the preconditioned animals, reflecting the less severe ultimate brain damage. Accordingly, the neuroprotection afforded to the preconditioned animals was not the result of any differences in the extent of anaerobic glycolysis, tissue acidosis, or depletion in high-energy reserves during hypoxia-ischemia but rather the result of other mechanisms that improved the metabolic status of the immature brain during the early hours of reperfusion following hypoxia-ischemia.

The Effect of Brief Post-Ischemic Hypothermia in Combination with Neuroprotective Agents on Hypoxic-Ischemic (H-I) Brain Damage in the Immature Rat • 1910

Literature in the immature animal indicates that the effect of mild to moderate hypothermia following an H-I insult is transient (Trescher et al, 1997), the protective effect being lost by 3-4 weeks of recovery. The objective of the present study was to determine, 1) the extent to which moderate vs severe H-I injury is ameliorated by post-ischemic hypothermia, and 2) the extent to which neuroprotective agents may be additive to the effects of post-ischemic hypothermia. To accomplish our first objective, 7-day postnatal rats were subjected to unilateral common carotid artery ligation and exposure to hypoxia in 8% oxygen while maintained at 37°C for periods of either 90 (moderate) or 180 (severe) minutes. Animals were then recovered for 3 hours in thermocontrolled waterbaths of 31, 34, or 37°C. Brains were examined neuropathologically at 7 or 21 days of recovery. At 7 days of recovery, only those animals which sustained a “moderate” H-I insult lasting 90 minutes, combined with post-ischemic hypothermia at 31°C displayed significantly less (p<0.05) brain damage then did their counterparts recovered at 34 or 37°C. By 21 days of recovery, this protective effect had dissipated. The findings indicate that post-ischemic mild hypothermia of 31°C can delay but not eliminate the extent of brain damage following a “moderate” (90 minute) H-I insult. Utilizing the results from our first investigation, 7-day rat pups were subjected to a moderate H-I insult at 37°C, afterwhich they immediately received subcutaneous injections of either, 1) 0.1 ml NS (control), 2) glutathione (1 mmol/kg), 3) N-acetylcysteine (6 mmol/kg), 4) flunarazine (30 mg/kg), 5) allopurinal (135 mg/kg), or 6) deferoxamine (100 mg/kg). Groups were then recovered at 31, 34, or 37°C for 3 hours, and then returned to their dams for neuropathologic assessment at 21 days of recovery. There was no difference in the extent of brain damage between control animals recovered at either 31, 34, or 37°C, confirming our earlier data. However, those animals receiving allopurinol combined with post-ischemic hypothermia of 31 or 34°C had significantly less (p<0.05) brain damage than did controls recovered at the same temperatures, as did those animals receiving glutathione in combination with hypothermia of 31°C during recovery. Thus, whereas mild post-ischemic hypothermia alone only delays brain damage following a moderate H-I insult, in combination therapy (allopurinol and glutathione) this important neuroprotective effect can be rendered permanent.

The effects of hypoxia-ischemia on expression of c-Fos, c-Jun and Hsp70 in the young rat hippocampus.

The expression of c-Fos, c-Jun and Hsp70 was examined in the hippocampus at 6, 12, 24, 48, 72 h, 4, 7 and 42 days following a combination of unilateral common carotid artery ligation and 60 min of systemic hypoxia (8% oxygen, 92% nitrogen) in 25-day-old male rats. While pyknotic cells were not visible in the hippocampus of control animals, pyknosis was evident in the ipsilateral, but not the contralateral hippocampus, of hypoxic-ischemic animals beginning at 24 h post-hypoxia. Immunohistochemical analysis revealed no c-Fos-, c-Jun- or Hsp70-immunoreactivity (IR) in any control animals. However, at 6 h post-hypoxia, Fos- and Jun-IR was evident throughout the injured ipsilateral hippocampus and later appeared throughout the contralateral hippocampus, which never showed signs of pyknosis. In contrast, Hsp70-IR was first observed at 24 h post-hypoxia and was restricted to the injured ipsilateral hippocampus. Hsp70-IR was not, however, limited to dying neurons. H-I/seizure animals did not express these proteins at any time point. These results suggest that, even in irreversibly injured neurons, Fos, Jun and Hsp70 appear to be involved in the aftermath of ischemia but probably do not play a pivotal role in the outcome of H-I compromised cells. Furthermore, compounded injury (H-I/seizure) appears to block the synthesis these proteins.

Influence of age on the cerebral lesions in an immature rat model of cerebral hypoxia–ischemia: a light microscopic study

The most frequently used model of neonatal cerebral hypoxia–ischemia consists of a 7-day postnatal rat model with combined common carotid artery ligation and hypoxemia. Neuropathologic studies have shown major differences between this 7-day postnatal rat model and a similar adult model in regard to overall cerebral vulnerability, type and distribution of lesions. It is not clear how and when during animals' development these changes in cerebral vulnerability take place. To determine this we studied groups of rats of 2 to 30 postnatal days. The animals underwent unilateral common carotid artery ligation followed by breathing in 8% oxygen for 30, 60, 90, or 120 min and their brains were examined at 24- or 72-h recovery intervals. Due to resistance of 2–3-day-old rats to develop cerebral hypoxic–ischemic damage, 5% O 2 was used instead of 8% O 2. The results indicate that: (i) There is an overall increase in severity of cerebral lesions on the side of common carotid artery ligation between 2 and 7 postnatal days. There is also an increase in the frequency of cerebral lesions in developing animals with increasing age. (ii) Hippocampus is remarkably resistant to hypoxic–ischemic insult at 2–3 postnatal days but becomes progressively vulnerable, and by age 13 postnatal days hippocampal vulnerability far exceeds that of cortex. (iii) Cortical lesions change from predominantly columnar cell death to laminar selective neuronal death at age 13 postnatal days. (iv) Also significant changes occur in relative vulnerability of various hippocampal regions during development. During the first 5 postnatal days relative vulnerability of hippocampal regions is similar, but as the animals' development proceeds and hippocampal vulnerability increases lesions tend to involve specific regions while sparing others. By age 13 postnatal days CA1 and lateral CA3 develop increased vulnerability while medial CA3 and fascia dentata become relatively resistant and by 21 postnatal days adult pattern of CA1 selective vulnerability is approached. The underlying mechanisms for these changes in regional vulnerability to cerebral hypoxia-ischemia during development should be sought in complex regional anatomic, functional, and metabolic alterations that take place as brain matures.

The effect of hyperglycemia on cerebral metabolism during hypoxia-ischemia in the immature rat.

Unlike adults, hyperglycemia with circulating glucose concentrations of 25-35 mM/L protects the immature brain from hypoxic-ischemic damage. To ascertain the effect of hyperglycemia on cerebral oxidative metabolism during the course of hypoxia-ischemia, 7-day postnatal rats underwent unilateral common carotid artery ligation followed by exposure to 8% O2 for 2 h at 37 degrees C. Experimental animals received 0.2 cc s.c. 50% glucose at the onset of hypoxia-ischemia, and 0.15 cc 25% glucose 1 h later to maintain blood glucose concentrations at 20-25 mM/L for 2 h. Control rat pups received equivalent concentrations or volumes of either mannitol or 1 N saline at the same intervals. The cerebral metabolic rate for glucose (CMRglc) increased from 7.1 (control) to 20.2 mumol 100 g-1 min-1 in hyperglycemic rats during the first hour of hypoxia-ischemia, 79 and 35% greater than the rates for saline-and mannitol-injected animals at the same interval, respectively (p < 0.01). Brain intracellular glucose concentrations were 5.2 and 3.0 mM/kg in the hyperglycemic rat pups at 1 and 2 h of hypoxia-ischemia, respectively; glucose levels were near negligible in mannitol- and saline-treated animals at the same intervals. Brain intracellular lactate concentrations averaged 13.4 and 23.3 mM/kg in hyperglycemic animals at 1 and 2 h of hypoxia-ischemia, respectively, more than twice the concentrations estimated for the saline- and mannitol-treated littermates. Phosphocreatine (PCr) and ATP decreased in all three experimental groups, but were preserved to the greatest extent in hyperglycemic animals. Results indicate that anaerobic glycolytic flux is increased to a greater extent in hyperglycemic immature rats than in normoglycemic littermates subjected to cerebral hypoxia-ischemia, and that the enhanced glycolysis leads to greater intracellular lactate accumulation. Despite cerebral lactosis, energy reserves were better preserved in hyperglycemic animals than in saline-treated controls, thus accounting for the greater resistance of hyperglycemic animals to hypoxic-ischemic brain damage.

Effect of Mild Hypothermia on Cerebral Energy Metabolism During the Evolution of Hypoxic-Ischemic Brain Damage in the Immature Rat

Intraischemic hypothermia (34 degrees C and 31 degrees C) has a profound neuroprotective effect on the brain of the immature rat. Hypothermia immediately after hypoxia-ischemia is not beneficial. To determine the mechanisms by which mild to moderate hypothermia affects cerebral energy metabolism of the brain of the newborn rat pup, we examined alterations in cerebral glycolytic intermediates and high-energy phosphate compounds during intraischemic and postischemic hypothermia and correlated these findings with known neuropathologic injury. Seven-day-old rat pups underwent unilateral common carotid artery ligation and exposure to hypoxia in 8% oxygen at either 37 degrees C, 34 degrees C, or 31 degrees C for 3.0 hours. Separate groups were exposed to hypoxia-ischemia at 37 degrees C for 3 hours but recovered at either 37 degrees C, 34 degrees C, or 31 degrees C. At 60, 120, and 180 minutes of intraischemic hypothermia and at 10, 30, 60, and 240 minutes of postischemic hypothermia, individual rat pups were quick-frozen in liquid nitrogen for later determination of cerebral concentrations of glucose, lactate, ATP, and phosphocreatine. Cerebral glucose was significantly higher and lactate significantly lower in the 31 degrees C animals during hypoxia-ischemia than either the 34 degrees C or 37 degrees C groups. Brain ATP concentrations were completely preserved during hypoxia-ischemia at 31 degrees C, whereas 34 degrees C of hypothermia had no effect on preserving high-energy phosphate compounds compared with those animals in the 37 degrees C group. Postischemic hypothermia of either 34 degrees C or 31 degrees C had no effect on the rate or extent of recovery of glycolytic intermediates or high-energy phosphate compounds compared with the normothermic 37 degrees C rat pups. Moderate hypothermia of 31 degrees C completely inhibits the depletion of ATP during hypoxia-ischemia, a mechanism that likely accounts for its neuroprotective effect. No preservation of ATP was seen, however, during intraischemic mild hypothermia of 34 degrees C despite the relatively profound neuroprotective effect of this degree of temperature reduction. Thus, the mechanisms by which mild hypothermia is neuroprotective are temperature dependent and may act at more than one point along the cascade of events eventually leading to hypoxic-ischemic brain damage in the immature rat.