CT perfusion-derived mean transit time of cortical brain has a negative correlation with the plasma level of Nitric Oxide after subarachnoid hemorrhage
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- Zheng, R., Qin, L., Li, S. et al. Acta Neurochir (2014) 156: 527. doi:10.1007/s00701-013-1968-6
Vasospasm of both large and small parenchymal arteries may contribute to the occurrence of delayed ischemic neurological deficits, and nitric oxide(NO) is an important mediators in the development of cerebral vasospasm after subarachnoid hemorrhage (SAH). We used a rabbit two-hemorrhage model to investigate changes in plasma NO after SAH, and the relationship between NO and brain microcirculation.
SAH was induced in rabbits and a control group was sham operated. There were 32 rabbits in each group that survived the second operation, and they were randomly assigned to four groups of eight rabbits each for follow-up assessments on Days 1, 4, 7, or 14, respectively. Cerebral blood flow (CBF), cerebral blood volume (CBV), and mean transit time (MTT) were calculated at six regions of interest (ROIs): symmetrical areas of the frontal, parietal-occipital, and temporal lobes. Before the contrast CT scan, blood was drawn from the central artery of the ear for measurement of plasma NO.
In the control group, there was no difference in CBV, CBF, and MTT in the six ROIs, and plasma NO was unchanged. Compared to controls, in the SAH group, CBV decreased slightly in the six ROIs (P > 0.05), frontal lobe CBF decreased, MTT increased (P < 0.05, for both), and NO plasma levels were significantly lower (P < 0.01).
There was a significant correlation between the increase in MTT and the decrease in plasma NO (P < 0.05), We hypothesized that normalization of NO might have a positive influence on brain microcirculation following SAH.
KeywordsSubarachnoidHemorrhage cerebralMicrocirculation nitricOxide cerebralBloodFlow cerebralBlood volume mean transitTime
Cerebral vasospasm (CVS) remains one of the main causes for poor clinical outcomes in patients after aneurysmal subarachnoid hemorrhage (SAH) . After a single SAH, CVS often occurs at three to four days, reaches its maximum incidence and severity between six and eight days, and usually resolves within 12 to14 days [1, 3]. Angiographic vasospasm occurs in 30 to70 % of patients with SAH, but leads to delayed, clinically evident ischemic neurological deficits in only 20-30 % of patients. About half of these patients suffer severe permanent neurological dysfunction or death . However, the disturbance of cerebral perfusion after SAH is caused not only by proximal artery segment vasospasm, but also by vasospasm of distal arteries and intraparenchymal arterioles .
Pennings et al., were the first to report direct visualization of the responses of the human cerebral microcirculation to hyperventilation using orthogonal polarization spectral imaging (OPS). They observed and quantified alterations in microvascular diameters, and found that arterioles responded to hyperventilation by contraction, whereas larger vessels were unaffected. SAH was associated with increased contractility of arterioles in patients undergoing early aneurysm surgery, as indicated by enhanced vasoconstriction in response to hyperventilation . Microcirculatory dysfunction related to endothelial damage, microvascular thrombosis, and loss of auto-regulation has been implicated in the pathogenesis of delayed cerebral ischemia [16, 19].
Nitric oxide (NO) is a mediator of vasomotor tone that may impact CV development after SAH. Endothelium-derived NO (eNO) inhibits platelet aggregation and adhesion, smooth muscle proliferation, endothelial cell apoptosis, and leukocyte adhesion to the endothelium. Decreased bioavailability of endogenous NO has, thus, been widely accepted as an important part of the pathogenesis of delayed CVS after SAH in humans. [2, 15, 21] .
Neuschmelting et al., reported that locally reduced NO availability in basilar arterial plasma is associated with CVS after SAH in acute animal experiment . Sabri et al., used a mouse model of SAH and the results suggest that CVS is associated with activation of eNOS in the brain tissue. This is accompanied by increased O2− and ONOO− but decreased NO, suggesting that there is eNOS uncoupling, which theoretically could contribute to secondary complications of SAH such as vasospasm and microthromboembolism and eventually, neuronal cell death. Pluta et al., reported that the subarachnoid blood scavenges NO and stimulates production of endogenous inhibitors of eNOS, leading to decreased NO and transient vasoconstriction after SAH .
The aim of our study was to assess changes in hemodynamic parameters in different regions of the brain after SAH, and to examine the relationship between hemodynamic parameters and the level of NO using a rabbit as an animal SAH model.
Materials and methods
Induction of subarachnoid hemorrhage
The ethics Subcommittee for Animal Care of the China Medical University approved the use of animals and all procedures followed their guidelines . A total of eighty-five adult New Zealand White rabbits (2.5–3.5 kg) of either sex weighing 2.5–3.5 kg were randomly assigned to two experimental groups, SAH group (n = 51), control group (n = 34) randomly. Eighty-five New Zealand white rabbits were initially anesthetized with 3 %–5 % isoflurane. Intravenous ketamine (3 mg/kg) and diazepam (0.3 mg/kg) were given to maintain anesthesia during intubation , SAH was induced in 51 rabbits and 34 rabbits were sham operated as controls. Thirty-two rabbits in each group survived the second operation and were randomly assigned to four groups of eight rabbits each for follow-up assessments on days 1, 4, 7, or 14, respectively. The SAH group was subjected to percutaneous puncture into the cisterna magna. There was 1.5 mL/kg of blood withdrawn from the central ear artery and then was injected back into the cisterna magna (3–5 minutes). The animals were placed in a positioning device in the prone position with the head secured at a downward angle of 30° to facilitate the formation of a blood clot around the basilar artery. After 48 hours, the identical operative procedure was repeated to induce SAH. In sham operated rabbits, 1.5 mL/kg of PBS solution was injected into the cistern magna instead of autologous blood. After 48 hours, the same procedure was done, but the volume of PBS solution was reduced to 1.0 mL/kg. All of the animals were awake within two hours after the operations [22, 25], similar to the results reported by the others.
The postoperative neurologic deficit of the animals was assessed every 24 hours using the scoring system reported by Strong et al. Posture, gait, and righting reflexes were each given a score: 0 = normal; 1 = mild; 2 = moderate; and 3 = severely impaired. Front and back reflexes were also scored: 0 = normal; 1 = brisk; 2 = spreading; and 3 = clonus. The sum of the individual observations provided the overall neurologic score. One individual, blinded to the study design, completed all neurologic assessments .
The whole brain perfusion method was used to study the hemodynamic parameters. The rabbits were anesthetized using intravenous pentobarbital (3 %, 30 mg/kg) injected through the auricular vein. The animals were placed in a prone position on a custom designed and custom built device with their heads secured stationary and parallel to the CT couch. To correct the position, a non-contrast CT head scan was performed before perfusion (80 kV, 100 mA, matrix 512 × 512, slice thickness 5 mm). For perfusion imaging, the length of the scan was 80 mm, and consisted of 16 5-mm sections. The contrast material (Omnipaque, 300 mg/mL; 1.5 mL/kg, ) was injected at a rate of 1 mL/s through the auricular vein with an automatic injector (Urich). Three hundred and twenty images were collected at each of the 16 section locations while the couch remained stationary. Scanning took 32 s to complete. The images were acquired at 0.5-s intervals and 100 kVp and 80 mA, 512 × 512 image matrix, 120-mm FOV, and 1.0 s per rotation [5, 20, 25].
Analysis of CBF, CBV and MTT
Functional maps were calculated using CT perfusion software (Philips, ICT 256). Arterial concentration curves were obtained from a 2 × 2 pixel region of interest (ROI) in the internal carotid artery in the section that showed the earliest arrival of contrast. We placed a 2 × 2 pixel ROI in the internal carotid artery on the section that showed the greatest area under the curve. The superior sagittal sinus was selected as the venous outflow function. Six ROIs were located symmetrically in the frontal, parietal–occipital, and temporal lobes in a band-like cortical of the maps. In order to verify the reliability and mean values of the result, we hand-drew two or three cines in every ROI and calculated the evaluation value. Thresholds were applied to limit the effects of large blood vessels on the tissue measurements (CBV > 8.0 mL/100 g; CBF > 250 mL/100 g per minute) .
After CT perfusion, animals were deeply anesthetized using intravenous ketamine (3 mg/kg), 150 mL PBS solution, and 500 mL 10 % formalin injected via the left ventricle over 30 minutes. Brain tissues located symmetrically in the frontal, parietal–occipital, and temporal lobes were collected and stored in 10 % buffered formalin [5, 12].
To confirm the presence and location of neuronal damage after SAH, we performed perfusion-fixation of the brain at 24 hours. Using a rabbit brain matrix, we cut 4 × 2 mm sections. Sections were paraffin-processed and microtome-sectioned into 4-μm thick sections before being stained with hematoxylin-eosin (H&E) to identify neuronal necrosis and apoptosis.
Nitric oxide content
We analyzed plasma NO using an enzyme-linked immunosorbent assay (ELISA, CUSABIO USA, Rabbit NO ELISA Kit). The sensitivity of the assay was 0.156 nmol/mL. All the assays were carried out in duplicate. The ELISA 96-well microtiter plates were analyzed using a microplate photometer with the maximum absorbance at 450 nm. Before the contrast CT scan, about 2 mL of arterial blood was withdrawn from the central artery of the ear with a sterile syringe. The blood samples were centrifuged at 3000 g for 15 minutes to collect about 1 mL of plasma, and stored at −80°Cuntil analysis.
SPSS statistical software version 17.0 (SPSS, Chicago, IL, USA) was used for analysis. Statistical significance between two means and multiple means was determined by parametric one-way ANOVA and T-testing. Multiple linear regression analysis was used to identify which CT perfusion parameters (CBF, CBV, or MTT) were significantly correlated with NO. Statistical significance was considered if the P-value was less than 0.05 (P < 0.05).
Changes in nitric oxide levels following SAH
Correlation of NO with CT perfusion
Vasospasm is a common complication that follows SAH, and leads to delayed ischemic neurological deficits in 20-30 % of those affected. According to Frontera et al., “DCI is a more clinically meaningful definition than either symptomatic deterioration alone or the presence of arterial spasm by angiography or TCD ” . A few reports have suggested disturbances of microcirculation after SAH aside from known infarct patterns of delayed CVS. Using orthogonal polarization imaging (OPS), Pennings et al., found that microvascular tonus was increased in patients undergoing aneurysm surgery within 48 hours after bleeding . In a study using magnetic resonance imaging (MRI), Weidauer et al., found focal laminar cortical infarcts after SAH . Others have found a significant correlation between MTT values and neurovascular findings. Wintermark et al., reported that MTT was significantly more sensitive, and CBF and CBV were significantly more specific . Kanazawa et al., found that patients with significant vasospasm had longer MTT compared with patients in whom vasospasm was negligible or absent . Kunze et al., found that repeatedly obaining PCT is a valuable screening tool to detect vasospasm and to decide whether a patient should be forwarded to angiography . Sanellia et al., in their results support the need for prospective clinical research focused on developing CTP as a prognostic tool for DCI and poor outcomes, which may provide valuable information in the management and treatment of aneurysmal SAH .
Multiple mechanisms might be involved in the maintenance and reversal of microcirculation in brain tissue after SAH. Alteration of the NO/NOS pathway is one of the molecular mechanisms that participate in brain injury after SAH. Other molecular mechanisms include endothelin-1 (ET-1) release, oxidative stress, inflammation, cell death pathways, and platelet activation and aggregation.
As a mediator of vasomotor tone, NO may impact CVS development after SAH . Endothelium-derived NO inhibits platelet aggregation and adhesion, smooth muscle proliferation, endothelial cell apoptosis, and leukocyte adhesion to the endothelium. Decreased bioavailability of endogenous NO has been widely accepted as an important factor in the pathogenesis of delayed CVS after SAH [11, 17, 21]. Pluta et al., reported that the subarachnoid blood scavenges NO and stimulates production of endogenous inhibitors of eNOS, leading to decreased NO and transient vasoconstriction after SAH .
Alexander et al., found that the endothelial NO synthase (eNOS) gene may play a role in the outcome following SAH. Decreased production of this gene leads to decrease NO formation and an environment favoring the development of CVS . In another study, wild-type mice were subjected to hypoxic preconditioning or normoxia followed 24 hours later by SAH. Hypoxic preconditioning prevented reductions in NO after SAH and increased NO levels in both sham-operated and SAH-operated mice . Five days after SAH induction in a one-hemorrhage rabbit model, other investigators found that local NO metabolites were lacking in basilar arterial plasma, and this was associated with CVS after SAH . In the current investigation, the levels of NO in plasma were decreased after and consistent with the findings of others. We were surprised to find that NO tended to increase over time in the 14-Day group, and was significantly different compared with the 4-Day and 7-Day groups. We hypothesize that regulation of NO-generated mechanisms plays a role on pathophysiology change of brain after SAH.
Jeon H et al., has described methods to create SAH in animal models . We found that the rabbit was easy to manipulate and operate. Zhou ML et al., pointed out that the two-hemorrhage model is more appropriate than the one-hemorrhage model for inducing SAH or cerebral vasospasm in rabbits since it produces more severe vasospasm with a low mortality and a time course of vasospasm similar to that in humans. Therefore, the two-hemorrhage model in rabbits has been widely accepted in the field for the investigation of the mechanisms of and therapeutic approaches for cerebral vasospasm . This, the rabbit double subarachnoid hemorrhage model is well documented, and shows a biphasic pattern of early and delayed vasospasm similar to that found in humans . In our experiment, the rabbits were allowed to breathe spontaneously and were kept in the head-down position. The mortality rates in our study corresponded with the results of other clinical and experimental studies.
On the other hand, there are some limitations of this study. The reasons of the death of rabbits remains unclear. We failed to observe the change of CTP and NO level in the same group at various time points. Leenders et al., has pointed out that that CTP parameters varied in a large range due to individual differences. Further studies are needed to clarify the specific mechanism of action and prove the efficacy of NO following SAH.
We found that MTT was the only CT perfusion parameter that was significantly negatively correlated with the level of NO in plasma at equivalent time points in the cortical brain after SAH. Increased MTT and decreased CBF changed significantly after SAH compared with the sham-operated group at the same time points. These findings indicate the time-dependent activation of pathological mechanisms that participate in brain injury after SAH, and lend support to the use of NO treatments after SAH to reduce the risk of poor outcomes. However, further studies are needed to prove the efficacy of NO following SAH.
This work was supported by grant 81071151 from the National Natural Science Foundation of China.
Conflicts of interest