Apoptotic Neuronal Death following Deep Hypothermic Circulatory Arrest in Piglets

Background: Deep hypothermic circulatory arrest (DHCA), as used in infant heart surgery, carries a risk of brain injury. In a piglet DHCA model, neocortical neurons appear to undergo apoptotic death. Caspases, cytochrome c, tumor necrosis factor (TNF), and Fas play a role in apoptosis in many ischemic models. This study examined the expression of these factors in a DHCA piglet model.

Methods: Thirty-nine anesthetized piglets were studied. After cardiopulmonary bypass (CPB) cooling of the brain temperature to 19[degrees]C, DHCA was induced for 90 min, followed by CPB rewarming. After separation from CPB, piglets were killed at 1, 4, 8, 24, and 72 h and 1 week. Caspase-8 and -3 activity, and concentrations of TNF-[alpha], Fas, Fas-ligand, cytochrome c, and adenosine triphosphate (ATP) were measured in the neocortex by enzymatic assay and Western blot analysis. Caspase-8 and -3 activity and cell death were examined histologically. Significance was set at P < 0.05.

Results: In neocortex, damaged neurons were not observed in control (no CPB), rarely observed in CPB (no DHCA), and rarely observed in the DHCA 1-h, 4-h, and 1-week reperfusion groups. However, they were seen frequently in the DHCA 8-, 24-, and 72-h reperfusion groups. Although neuronal death was widespread 8-72 h after DHCA, cortical ATP concentrations remained unchanged from control. Both caspase-3 and -8 activities were significantly increased at 8 h after DHCA, and caspase-3 concentration remained elevated for as long as 72 h. Caspase-3 and -8 activity was also observed in damaged neocortical neurons. Cytosolic cytochrome c and Fas were significantly expressed at 1 h and 4 h after DHCA, respectively. Fas-ligand and TNF-[alpha] were not observed in any group.     

Hypothermia at 10°C Reduces Neurologic Injury After Hypothermic Circulatory Arrest in the Pig

Abstract    Background: We have previously reported that sensory, motor neocortex, and hippocampus are selectively vulnerable to injury in an acute porcine model of HCA at 18°C. This study was undertaken to assess whether further cooling to 10°C can reduce neurological injury during HCA. Methods: Twelve piglets underwent 75 minutes of HCA at 18°C (n = 6) and 10°C (n = 6). Four served as normal controls. After gradual rewarming and 80 minutes of reperfusion, treatment animals were sacrificed and brains were perfusion-fixed and cryopreserved. Regional patterns of neuronal apoptosis after HCA were characterized by in situ DNA fragmentation using TUNEL histochemistry. Hematoxylin and eosin histology was used to characterize cell damage morphologically. TUNEL-positive cells were scored on a scale of 0 to 5. Grade 0: no TUNEL-positive cells; Grade 1: < 10%; Grade 2: 10% to 25%, Grade 3: 25% to 50%, Grade 4: 50% to 75%; and Grade 5: > 75%. Results: TUNEL-positive cells indicating DNA fragmentation were scored in the motor and sensory neocortex, hippocampus, cerebellum, thalamus, and medulla of animals treated with 18°C and 10°C HCA and were significantly greater than in normal controls. Profound cooling to 10°C resulted in a significant reduction of neuronal injury in the neocortex and hippocampus. Conclusions: This data support that cerebral protection may be better at very cold temperatures compared to 18°C hypothermia. Regions selectively vulnerable to neuronal injury are offered more neural protection by profound hypothermia. These affects are observed in the acute state, suggesting activation of the apoptotic mechanisms at early stages can be inhibited by profound hypothermia.     

Roles of Neuronal Nitric Oxide Synthase and Inducible Nitric Oxide Synthase in Intestinal Transplantation of Rats

Objective

The study was designed to evaluate the role of neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) in ischemia-reperfusion injury (IRI) and acute rejection (AR) in rat intestinal transplantation, by administration of nitric oxide inhibitor N^G-nitro-L-arginine methyl ester (LNAME).

Materials and methods

Rats that underwent orthotopic intestinal transplantation were assigned to 2 sets of groups: (1) iso-geneic group (Lewis-Lewis), L-NAME 0 mg/kg/d group (1-1), 4 mg/kg/d (group 1-2), or 8 mg/kg/d (group 1-3) injected intraperitoneally or (2) allogeneic group (Dark Agouti-Lewis), L-NAME 0 mg/kg/d (group 2-1) or 8 mg/kg/d (group 2-2) injected intraperitoneally. We examined survival times, light microscopy as well as maltose absorption tests. The nNOS and iNOS activities were measured by immunohistochemical methods.

Results

Histologic examination showed inhibited iNOS activity compared with group l-l, and Park scores decreased significantly in group 1-2 at 30 minutes after reperfusion (1.42 ± 0.38 vs 2.58 ± 0.49, P < .01). Both iNOS and nNOS activities were inhibited and Park scores increased significantly in group 1-3 from 30 minutes to day 3 after reperfusion (P < .0l). nNOS activity decreased and iNOS activity increased among group 2-1 during AR. Compared with group 2-1, iNOS activity was inhibited, progression of AR delayed, and survival significantly prolonged in group 2-2 (10.17 ± 0.98 vs 6.83 ± 0.75, P < .01).

Conclusion

This study suggested that decreased nNOS and increased iNOS activity both contributed to IRI and AR. More importantly, nNOS more importantly than iNOS activity was closely related to graft structure and function.     

Neuronal Nitric Oxide Synthase Mediates Halothane-induced Cerebral Microvascular Dilation

Background: The causes of volatile anesthetic-induced cerebral vasodilation include direct effects on smooth muscle and indirect effects via changes in metabolic rate and release of mediators from vascular endothelium and brain parenchyma. The role of nitric oxide and the relative importance of neuronal and endothelial nitric oxide synthase (nNOS and eNOS, respectively) are unclear.

Methods: Rat brain slices were superfused with oxygenated artificial cerebrospinal fluid. Hippocampal arteriolar diameters were measured using computerized videomicrometry. Vessels were preconstricted with prostaglandin F2[alpha] (PGF2[alpha]; halothane group) or pretreated with 7-nitroindazole sodium (7-NINA, specific nNOS inhibitor, 7-NINA + halothane group) or N-nitro-L-arginine methylester (L-NAME; nonselective NOS inhibitor, L-NAME + halothane group) and subsequently given PGF2[alpha] to achieve the same total preconstriction as in the halothane group. Increasing concentrations of halothane were administered and vasodilation was calculated as a percentage of preconstriction.

Results: Halothane caused significant, dose-dependent dilation of hippocampal microvessels (halothane group). Inhibition of nNOS by 7-NINA or nNOS + eNOS by L-NAME similarly attenuated halothane-induced dilation at 0.6, 1.6, and 2.6% halothane. The dilation (mean +/- SEM) at 1.6% halothane was 104 +/- 10%, 65 +/- 6%, and 51 +/- 9% in the halothane, 7-NINA + halothane and L-NAME + halothane groups, respectively. The specificity of 7-NINA was confirmed by showing that acetylcholine-induced dilation was not inhibited by 7-NINA but was converted to constriction by L-NAME.     

Hypothermic Asanguineous Circulatory Arrest in Adult Dogs

A new technique for the institution of hypothermic asanguineous circulatory arrest (HACA) is described and evaluated. It employs rapid cooling and hemodilution at high flow rates. Survival and protection of neurologic and other organ function were obtained using asanguineous circulatory arrest, and the method appears to be an improvement over circulatory arrest using conventional methods for cooling. The total duration of extracorporeal circulation is much shorter than with currently used methods of circulatory arrest. Applications of this technique in general and transplantation surgery as well as cardiac surgery are discussed.     

Carotid Artery Doppler Flow Pattern After Deep Hypothermic Circulatory Arrest in Neonatal Piglets

The mechanisms of cerebral injury after cardiac surgery in neonates are not clear. The aim of the study was the analysis of flow changes in the carotid artery of neonatal piglets after deep hypothermic circulatory arrest (DHCA). Eight neonatal piglets were connected to cardiopulmonary bypass (CPB) and underwent (i) cooling to 18°C core temperature within 30 min, (ii) DHCA for 90 min, and finally (iii) rewarming to 37°C after cross‐clamp release (60 min of reperfusion). The blood flow was measured in the left carotid artery by an ultrasonic flow probe before CPB (baseline; T₀), immediately after termination of reperfusion on CPB (T₁), 30 min later (T₂), and 60 min later (T₃). Additionally, the pulsatility index and the resistance index were calculated and compared. Finally, the relationship between the carotid artery flow and the corresponding pressure at each time‐point was compared. After termination of CPB (T₁), the mean carotid artery flow was reduced from 45.26 ± 2.58 mL/min at baseline to 23.29 ± 2.58 mL/min (P < 0.001) and remained reduced 30 and 60 min later (P < 0.001 vs. baseline). Both the pulsatility index and the resistance index were increased after termination of reperfusion, with the maximum occurring 30 min after CPB end. In conclusion, the carotid artery Doppler flow in neonatal piglets was reduced after DHCA, while the indices of pulsatility and resistance increased.     

Cerebral Consequences of Hypothermic Circulatory Arrest in Adults

Despite widespread use of hypothermic circulatory arrest (HCA) in aneurysm surgery and for repair of congenital heart defects, there is continued concern about possible adverse cerebral sequelae. The search for ways to improve implementation of HCA has inspired retrospective clinical studies to try to identify risk factors for cerebral injury, and clinical and laboratory investigations to explore the physiology of HCA. At present, risk factors associated with less favorable cerebral outcome after HCA include: prolonged duration of HCA (usually greater than 60 min); advanced patient age; rapid cooling (less than 20 min); hyperglycemia either before HCA or during reperfusion; preoperative cyanosis or lack of adequate hemodilution; evidence of increased oxygen extraction before HCA or during reperfusion; and delayed reappearance of electroencephalogram (EEG) or marked EEG abnormality. Strategies advocated to increase safety of HCA include: pretreatment with barbiturates and steroids; use of alpha-stat pH regulation during cooling and rewarming; intraoperative monitoring of EEG; slow and adequate cooling, including packing of the head in ice; monitoring of jugular venous oxygen content; hemodilution; and avoidance of hyperglycemia. Current investigation focuses on delineating the relationship of cerebral blood flow (CBF) to cerebral oxygen consumption and glucose metabolism during cooling, HCA, rewarming, and later recovery, and identifying changes in acute intraoperative parameters, including the presence of intracerebral enzymes in cerebral spinal fluid, with cerebral outcome as assessed by neurological evaluation, quantitative EEG, and postmortem histology. Clinically, intraoperative monitoring of EEG and measurement of CBF by tracer washout or Doppler flows are contributing to better understanding of the physiology of HCA, and in the laboratory, nuclear magnetic resonance (NMR) spectroscopy has provided valuable insights into the kinetics of intracerebral energy metabolism. Promising strategies for the future include investigation of other pharmacological agents to increase cerebral protection, and use of "cerebroplegia" or intermittent perfusion between intervals of HCA to improve cerebral tolerance for longer durations of HCA.     

Deep Brain Hyperthermia While Rewarming from Hypothermic Circulatory Arrest

Abstract   Background: Neurologic injury is a feared and serious long-term complication of cardiopulmonary bypass (CPB) and deep hypothermic circulatory arrest (DHCA). Postoperative hyperthermia was found to enhance postischemic neurologic injury. The use of core temperature as the reference point through CPB assumes parallel changes in brain temperature. We tested the hypothesis that regional and deep brain temperature (DBT) differ during cooling, DHCA, and rewarming. Methods: Neonatal piglets (n = 9) were subject to CPB and cooled to rectal temperature (RT) of 18 °C, 30 minutes of DHCA were initiated, and subsequently the piglets were rewarmed to RT of 36.5 °C and weaned from CPB. Temperature probes were inserted into the DBT targeting the caudate and thalamic nuclei, their position confirmed by pathology. Superficial brain temperature was measured by a temperature probe inserted extradurally. RT, nasopharyngeal (NPT), and tympanic (TT) temperatures were recorded. Results: During cooling the deep brain cooled faster and to lower temperatures compared to RT and TT; NPT reflected DBT accurately. During rewarming DBT was significantly higher than RT and TT. By the end of rewarming the difference between the deep brain and the RT reached statistical significance (30 minutes: 35.1 ± 0.7 vs. 32.3 ± 0.7 p < 0.05, respectively, 40 minutes: 37.5 ± 0.3 vs. 34.7 ± 0.8 p < 0.05, respectively). Conclusion: Deep brain hyperthermia routinely occurs during the last stages of rewarming following DHCA. DBT is accurately reflected by NPT and is directly correlated with inflow temperature. Therefore, during rewarming inflow temperatures should not exceed 36 °C and NPT should be closely monitored.     

Isoflurane-induced Cerebral Hyperemia in Neuronal Nitric Oxide Synthase Gene Deficient Mice

Background: Nitric oxide (NO) has been reported to play an important role in isoflurane-induced cerebral hyperemia in vivo. In the brain, there are two constitutive isoforms of NO synthase (NOS), endothelial NOS (eNOS), and neuronal NOS (nNOS). Recently, the mutant mouse deficient in nNOS gene expression (nNOS knockout) has been developed. The present study was designed to examine the role of the two constitutive NOS isoforms in cerebral blood flow (CBF) response to isoflurane using this nNOS knockout mouse.

Methods: Regional CBF (rCBF) in the cerebral cortex was measured with laser-Doppler flowmetry in wild-type mice (129/SV or C57BL/6) and nNOS knockout mice during stepwise increases in the inspired concentration of isoflurane from 0.6 vol% to 1.2, 1.8, and 2.4 vol%. Subsequently, a NOS inhibitor, Nomega -nitro-L-arginine (L-NNA), was administered intravenously (20 mg/kg), and 45 min later, the rCBF response to isoflurane was tested again. In separate groups of wild-type mice and the knockout mice, the inactive enantiomer, Nomega -nitro-D-arginine (D-NNA) was administered intravenously in place of L-NNA. Brain NOS activity was measured with radio-labeled L-arginine to L-citrulline conversion after treatment with L-NNA and D-NNA.

Results: Isoflurane produced dose-dependent increases in rCBF by 25 +/- 3%, 74 +/- 10%, and 108 +/- 14% (SEM) in 129/SV mice and by 32 +/- 2%, 71 +/- 3%, and 96 +/- 7% in C57BL/6 mice at 1.2, 1.8, and 2.4 vol%, respectively. These increases were attenuated at every anesthetic concentration by L-NNA but not by D-NNA. Brain NOS activity was decreased by 92 +/- 2% with L-NNA compared with D-NNA. In nNOS knockout mice, isoflurane increased rCBF by 67 +/- 8%, 88 +/- 12%, and 112 +/- 18% at 1.2, 1.8, and 2.4 vol%, respectively. The increase in rCBF at 1.2 vol% was significantly greater in the nNOS knockout mice than that in the wild-type mice. Administration of L-NNA in the knockout mice attenuated the rCBF response to isoflurane at 1.2 and 1.8 vol% but had no effect on the response at 2.4 vol%.     

Inhibition of Nitric Oxide Synthase Prevents Hyporesponsiveness to Inhaled Nitric Oxide in Lungs from Endotoxin-challenged Rats

Background: Inhalation of nitric oxide (NO) selectively dilates the pulmonary circulation and improves arterial oxygenation in patients with adult respiratory distress syndrome (ARDS). In approximately 60% of patients with septic ARDS, minimal or no response to inhaled NO is observed. Because sepsis is associated with increased NO production by inducible NO synthase (NOS2), the authors investigated whether NOS inhibition alters NO responsiveness in rats exposed to gram-negative lipopolysaccharide (LPS).

Methods: Sprague-Dawley rats were treated with 0.4 mg/kg Escherichia coli 0111:B4 LPS with or without dexamethasone (inhibits NOS2 gene expression; 5 mg/kg), L-NAME (a nonselective NOS inhibitor; 7 mg/kg), or aminoguanidine (selective NOS2 inhibitor; 30 mg/kg). Sixteen hours after LPS treatment, lungs were isolated-perfused; a thromboxane-analog U46619 was added to increase pulmonary artery pressure (PAP) by 5 mmHg, and the pulmonary vasodilator response to inhaled NO was measured.

Results: Ventilation with 0.4, 4, and 40 ppm NO decreased the PAP less than in lungs of LPS-treated rats (0.75 +/- 0.25, 1.25 +/- 0.25, 1.75 +/- 0.25 mmHg) than in lungs of control rats (3 +/- 0.5, 4.25 +/- 0.25, 4.5 +/- 0.25 mmHg; P < 0.01). Dexamethasone treatment preserved pulmonary vascular responsiveness to NO in LPS-treated rats (3.75 +/- 0.25, 4.5 +/- 0.25, 4.5 +/- 0.5 mmHg, respectively; P < 0.01 vs. LPS, alone). Responsiveness to NO in LPS-challenged rats was also preserved by treatment with L-NAME (3.0 +/- 1.0, 4.0 +/- 1.0, 4.0 +/- 0.75 mmHg, respectively; P < 0.05 vs. LPS, alone) or aminoguanidine (1.75 +/- 0.25, 2.25 +/- 0.5, 2.75 +/- 0.5 mmHg, respectively; P < 0.05 vs. LPS, alone). In control rats, treatment with dexamethasone, L-NAME, and aminoguanidine had no effect on inhaled NO responsiveness.