TY - JOUR
AU - Rungatscher,, Alessio
AB - Abstract Open in new tabDownload slide Open in new tabDownload slide OBJECTIVES Among the factors that could determine neurological outcome after hypothermic circulatory arrest (HCA) rewarming is rarely considered. The optimal rewarming rate is still unknown. The goal of this study was to investigate the effects of 2 different protocols for rewarming after HCA on neurological outcome in an experimental animal model. METHODS Forty-four Sprague Dawley rats were cooled to 19 ± 1°C body core temperature by cardiopulmonary bypass (CPB). HCA was maintained for 60 min. Animals were randomized to receive slow (90 min) or fast (45 min) assisted rewarming with CPB to a target temperature of 35°C. After a total of 90 min of reperfusion in both groups, brain samples were collected and analysed immunohistochemically and with immunofluorescence. In 10 rats, magnetic resonance imaging was performed after 2 and after 24 h to investigate cerebral perfusion and cerebral oedema. RESULTS Interleukin 6, chemokine (C-C motif) ligand 5, intercellular adhesion molecule 1 and tumour necrosis factor α in the hippocampus are significantly less expressed in the slow rewarming group, and microglia cells are significantly less activated in the slow rewarming group. Magnetic resonance imaging analysis demonstrated better cerebral perfusion and less water content in brains that underwent slow rewarming at 2 and 24 h. CONCLUSIONS Slow rewarming after HCA might be superior to fast rewarming in neurological outcome. The present experimental study demonstrated reduction in the inflammatory response, reduction of inflammatory cell activation in the brain, enhancement of cerebral blood flow and reduction of cerebral oedema when slow rewarming was applied. Hypothermic circulatory arrest, Rewarming, Cardiopulmonary bypass, Cerebral perfusion, Neurological outcome INTRODUCTION In aortic surgery, the requirement of interruption of blood flow through cardiopulmonary bypass (CPB) during aortic arch reconstruction causes diffuse ischaemic hypoxic injury to all body organs [1]. Hypothermic circulatory arrest (HCA) has been the surgical technique responsible for cerebral protection during aortic arch surgery. With the introduction of antegrade cerebral perfusion, many surgeons reduced the depth of hypothermia during HCA. In current clinical practice, HCA is performed at a temperature between 18°C and 19°C [2], but the evidence to support any range of temperature is controversial [3, 4]. Indeed, optimal temperature management and the ideal cerebral perfusion technique are still matters of debate. The occurrence of neurological damage, whether temporary or permanent, varies in different clinical studies (5–20%) and depends on the length of circulatory arrest, the temperature maintained during surgery, the rewarming rate and surgeon or institutional preferences [5]. Among the factors that could affect neurological outcomes, rewarming is considered less often, even though some clinical reports have pointed out the possible risk associated with rewarming [6]. A higher rewarming rate could rapidly reduce the adverse effects of hypothermia and CPB; on the other hand, a slow rewarming rate influence the duration of CPB and may exert negative effects on coagulation and infections. Patients who underwent fast rewarming had a higher incidence of cognitive deficits at 6 weeks [7]. Inflammation and apoptosis are the most relevant pathways activated in the brain after HCA. The chemokines (C-X-C motif) ligand 9, (C-X-C motif) ligand 10, (C-C motif) ligand 2 and nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) are the most upregulated genes. Concomitantly, genes involved in cell proliferation, cell–cell adhesion and structural integrity were significantly downregulated in the HCA-treated group [8]. The Society of Thoracic Surgeons, the Society of Cardiovascular Anaesthesiologists and the American Society of Extracorporeal Technology have released guidelines for temperature management during CPB [9]. Most of the recommendations are derived from expert opinion with a lack of any randomized studies. Indeed, although numerous studies are available on the neuroprotective effects of hypothermia, the impact of the heating mode has not yet been clarified. The goal of this preclinical observational study was to determine whether different rewarming rates (fast or slow) could have different neurological outcomes in terms of inflammatory activation, oxidative stress, cerebral perfusion and oedema after HCA. METHODS All experiments were performed in adherence with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (National Institutes of Health Publication No. 85-23, revised 1996). The protocol was approved by the University of Verona authority for experimental research. Experiments took place at the Interdepartmental Center for Research on Laboratory Animals of Biological Institutes, University of Verona, after approval from the Technical Scientific Committee and Ministry of Health (Registration Number 56DC9.32, 27 March 2018). A total of 44 male Sprague Dawley rats weighing 400 ± 50 g, aged 6–7 months, were used for the experiments. Surgical procedure After induction of anaesthesia with Sevoflurane 6% (Forene; Abbott, Baar, Switzerland), rats underwent tracheal intubation and ventilation by mechanical respirator for rodents (Harvard Model 687, Harvard Apparatus, Holliston, MA, USA) with a mixture of oxygen and anaesthetic Sevoflurane 2%, a 10-ml/kg tidal volume and a respiratory frequency of 80 breaths per minute. Ketoprofen 2 mg/kg was injected subcutaneously to maintain analgesia. During the surgical procedure, the temperature of the room was maintained between 23° and 25°C. An electrocardiographic monitor was positioned, and body temperature was monitored by a rectal probe and a probe inserted in the right jugular vein. A miniaturized 2-Fr diameter catheter (model SPR 838, Millar Instruments, Houston, TX, USA) was inserted into the right femoral artery to monitor systemic arterial pressure. The right femoral vein was cannulated, and 2 mg/kg of pancuronium-bromide was infused to obtain muscle relaxation. The animals were prepared for CPB as previously described [10, 11]. In brief, after isolation of the right external jugular vein, a 5-Fr diameter cannula was inserted and advanced in the right atrium, and 500 IU/kg of heparin was administered. The right common carotid artery was cannulated with a 24-G catheter, advanced to the aortic arch and then connected to the circuit arterial perfusion line. The extracorporeal circulation circuit comprised an outflow tube connected to the jugular vein, a venous reservoir connected to a vacuum (maintained between −40 and −60 cm H2O), a connection tube that runs through a roller pump (Stockert SIII, Sorin, Germany) and ends in an hollow fibre oxygenator with industrial standard characteristics (Sorin, Mirandola, Modena, Italy), and an inflow tube connected to the right carotid artery. The total length of the extracorporeal circuit was 80 cm; the outflow tube had 2 mm inside diameter whereas the remaining circuit had a 1.6-mm inside diameter; the total filling volume was 6 ml and comprised 50% lactated Ringer’s solution and 50% colloid solution (Voluven, Fresenius Kabi Italia Srl, Verona, Italy). The oxygenator exchange surface area was 450 cm2. A flow rate of 120–140 ml/kg/min and a mean arterial pressure range of 70–90 mmHg were assured, adjusting the vacuum and adding some priming volume if necessary, in order to maintain a haemodynamic balance. The temperature was adjusted by a heat exchanger (Sarns cardioplegia set, Terumo Cardiovascular System Corp., Ann Arbor, MI, USA) incorporated in the circuit, and it was modulated using intravascular temperature monitoring (ADInstruments, Oxford, UK). Experimental protocol Deep hypothermia (18–20°C) was induced with the help of the heat exchanger. CPB and the ventilator were turned off, and the rats were left in a state of HCA. After 60 min, CPB was re-established. The animals were randomized into 2 groups based on different rewarming durations. In the slow group (n = 22), the final temperature of 35°C was reached in 90 min. A 5°C temperature gradient between the jugular and the heat exchanger was maintained (Fig. 1). Figure 1: Open in new tabDownload slide The experimental study protocol comprised 30 min of cooling with CPB, 60 min of HCA and 90 min of reperfusion and rewarming. In the fast group, rewarming was achieved in 45 min, in the slow group, in 90 min. Arterial samples were collected for blood gas analysis before the procedure, after HCA, in the middle of the rewarming phase and at the end of the experiment. At the end of the protocol, brains were collected in paraformaldehyde to perform immunochemical and immunofluorescence analyses. In a subset of rats, MRI was performed at 2 and 24 h after the end of the experiment. CCL5: chemokine (C-C motif) ligand 5; CPB: cardiopulmonary bypass; HCA: hypothermic circulatory arrest; Iba1: ionized calcium-binding adapter molecule 1; ICAM-1: intercellular adhesion molecule 1; IL-6: interleukin 6; MDA: malondialdehyde; MRI: magnetic resonance imaging; TNFα: tumour necrosis factor α. Figure 1: Open in new tabDownload slide The experimental study protocol comprised 30 min of cooling with CPB, 60 min of HCA and 90 min of reperfusion and rewarming. In the fast group, rewarming was achieved in 45 min, in the slow group, in 90 min. Arterial samples were collected for blood gas analysis before the procedure, after HCA, in the middle of the rewarming phase and at the end of the experiment. At the end of the protocol, brains were collected in paraformaldehyde to perform immunochemical and immunofluorescence analyses. In a subset of rats, MRI was performed at 2 and 24 h after the end of the experiment. CCL5: chemokine (C-C motif) ligand 5; CPB: cardiopulmonary bypass; HCA: hypothermic circulatory arrest; Iba1: ionized calcium-binding adapter molecule 1; ICAM-1: intercellular adhesion molecule 1; IL-6: interleukin 6; MDA: malondialdehyde; MRI: magnetic resonance imaging; TNFα: tumour necrosis factor α. In the fast group (n = 22), a final temperature of 35°C was reached in 45 min by setting the temperature of the heat exchanger initially with 10°C of gradient; then, when the temperature of the jugular vein was 30°C, the gradient was lowered to 5°C. The rats were kept at this temperature until 90 min of reperfusion had been completed (Fig. 1). A subset of 18 rats was designated for magnetic resonance imaging (MRI). The rats were anaesthetized by inhalation of a mixture of oxygen and air containing 2–3% Sevoflurane; images were obtained using Bruker Biospec (Bruker Corporation, Billerica, MA, USA) tomographic equipment operating at 4.7 T. At the end of the reperfusion period, these animals were weaned from the bypass, the vascular access equipment was removed, the neck incision was sutured and Ketoprofen 2 mg/kg was injected subcutaneously. Protamine was not used to reverse the effects of heparin. Sevoflurane administration was stopped, and the rats were extubated. All 18 rats were successfully weaned from the ventilator; no relevant neurological damage was evident. After 1 h of observation, the rats underwent a second MRI brain scan. During the acquisition of the images, 1 rat from the fast rewarming group died of respiratory failure. An additional 7 rats died during the next few hours (4 in the slow rewarming group and 3 in the fast rewarming group) of respiratory distress and pulmonary oedema. These animals were excluded from the study. The remaining 10 rats underwent a third brain MRI analysis at 24 h after the end of the operation. After that, all rats were sacrificed using Sevoflurane overdoses. Arterial blood gas analysis Arterial samples were collected for blood gas analysis (pH, pO2, pCO2, haematocrit, lactate levels) before the procedure, after HCA, in the middle of the rewarming phase and at the end of the experiment. Tissue analysis A 24-G cannula was inserted in the right carotid artery towards the brain; 10 ml of phosphate-buffered saline was injected, followed by 10 ml of paraformaldehyde. A posterior craniotomy was performed, and the whole brain was removed and placed in paraformaldehyde. Ten brains (5 in the slow and 5 in the fast group) were used for immunochemical analysis. Tissue sections were observed with an Olympus BX51 optical microscope (Olympus, Tokyo, Japan) equipped with a digital camera (DKY-F58 CCD, JVC, Yokohama, Japan). Electronic images were analysed and processed using Image-ProPlus 7.0 software (Media Cybernetics, Silver Spring, MD, USA). Antibodies used to check the inflammatory response were specific for tumour necrosis factor α (TNF α) (Novus, Milan, Italy), interleukin 6 (IL-6) (Novus, Milan, Italy), chemokine (C-C motif) ligand 5 (CCL5) (Bioss, Woburn, MA, USA), intercellular adhesion molecule 1 (Bioss) and to check oxidative stress, antibodies for malondialdehyde (MDA) (Abcam, Cambridge, UK) were used. For immunofluorescence analysis, an additional 10 brains were analysed (5 in the slow group and 5 in the fast group). Tissue sections were incubated with anti-ionized calcium-binding adapter molecule 1 (Iba1) primary antibodies (Wako Chemicals, Richmond, VA, USA). This antibody is specific for microglia cell determinants of the first and primary immune defence of the central nervous system [12, 13]. After that, slides were incubated with carbocyanine monomer nucleic acid stain (TOPRO-3, molecular probes, Thermo Fisher Scientific, Waltham, MA, USA) (1:3000) for staining the nuclei. The tissue sections were evaluated using ImageJ software (US National Institutes of Health) under blinded conditions. A detailed process for the preparation of the brain sections is given in the Supplementary Material. In a subset of rats (3 slow group; 3 fast group), the brain wet/dry ratio was determined. The brains were removed whole and weighed immediately to obtain the initial weight (wet). Later, the brains were placed in a laboratory oven with a constant temperature of 105°C to allow the slow vaporization of water from the brain tissue. After 24 h, the brains were weighed again (dry), and the comparison between the 2 weights provided the wet/dry ratio, the index of tissue oedema. Magnetic resonance imaging Ten rats were subjected to MRI analysis 24 h after the operation. The data were exported to a workstation, registered to a template through FSL (Functional MRI of the brain Software Library) [14]. Perfusion maps were calculated according to the formula [(SIpost-SIpre)/SIpre] (where SI = signal intensity and pre-/post-suffixes refers to pre- and post-contrast agent injection). The values obtained are expressed as negative numbers; the lower the value, the higher is the degree of perfusion. Mean diffusivity maps were calculated using the built-in software of the MR imager (Paravision 5.1, Bruker). A detailed protocol for MRI analysis is given in the Supplementary Material. Statistical analyses Data analyses were performed using SPSS software version 21 (SPSS Inc., Chicago, IL, USA). The mean ± the standard deviation was calculated for all measurements. For tissue analysis, the average percentages of inflammatory markers, MDA and Iba1 expression were quantified in randomly selected brain regions comprising the cortex, the subventricular zone and the hippocampus. The number of Iba1 positive pixels was normalized to the total number of pixels of the regions of interest considered (250 images were quantified across the different conditions). The Student’s t-test or the Mann–Whitney non-parametric test was applied to compare the 2 groups; a P-value <0.05 was considered statistically significant. RESULTS Arterial blood gas analysis Blood gas analysis showed an increase in lactates during HCA, followed by a gradual normalization during rewarming. The pH returned to normal during rewarming, after the development of acidosis in the circulatory arrest phase. No substantial differences were identified between the fast and the slow groups (Table 1). Table 1: Blood gas analysis . . . . Slow . Fast . Slow . Fast . . Basal . 18°C . HCA . 45′ CPB . 45′ CPB . 90′ CPB . 90′ CPB . pH 7.39 ± 0.04 7.37 ± 0.04 6.94 ± 0.35 7.4 ± 0.03 7.5 ± 0.03 7.39 ± 0.03 7.38 ± 0.03 pCO2 (mmHg) 37 ± 2.5 34 ± 3 ≥115 23 ± 5.3 21 ± 3.7 31 ± 5.2 30 ± 5.8 pO2 (mmHg) 150 ± 10 165 ± 20 14 ± 5 447 ± 50 437 ± 50 213 ± 43 213 ± 43 Hct (%) 45 ± 2.5 47.6 ± 2.3 55 ± 3.3 27 ± 2.5 27 ± 2.5 25 ± 2.8 25 ± 2.8 Lat (mmol/l) 0.7 ± 0.2 1.8 ± 0.7 7.8 ± 2.5 8.7 ± 2.2 8.5 ± 2.4 6.1 ± 1.4 5.1 ± 2 . . . . Slow . Fast . Slow . Fast . . Basal . 18°C . HCA . 45′ CPB . 45′ CPB . 90′ CPB . 90′ CPB . pH 7.39 ± 0.04 7.37 ± 0.04 6.94 ± 0.35 7.4 ± 0.03 7.5 ± 0.03 7.39 ± 0.03 7.38 ± 0.03 pCO2 (mmHg) 37 ± 2.5 34 ± 3 ≥115 23 ± 5.3 21 ± 3.7 31 ± 5.2 30 ± 5.8 pO2 (mmHg) 150 ± 10 165 ± 20 14 ± 5 447 ± 50 437 ± 50 213 ± 43 213 ± 43 Hct (%) 45 ± 2.5 47.6 ± 2.3 55 ± 3.3 27 ± 2.5 27 ± 2.5 25 ± 2.8 25 ± 2.8 Lat (mmol/l) 0.7 ± 0.2 1.8 ± 0.7 7.8 ± 2.5 8.7 ± 2.2 8.5 ± 2.4 6.1 ± 1.4 5.1 ± 2 Non-significant differences in inter-group comparisons were found during the rewarming period (P > 0.05). CPB: cardiopulmonary bypass; HCA: hypothermic circulatory arrest; Hct: haematocrit; Lat: lactate. Open in new tab Table 1: Blood gas analysis . . . . Slow . Fast . Slow . Fast . . Basal . 18°C . HCA . 45′ CPB . 45′ CPB . 90′ CPB . 90′ CPB . pH 7.39 ± 0.04 7.37 ± 0.04 6.94 ± 0.35 7.4 ± 0.03 7.5 ± 0.03 7.39 ± 0.03 7.38 ± 0.03 pCO2 (mmHg) 37 ± 2.5 34 ± 3 ≥115 23 ± 5.3 21 ± 3.7 31 ± 5.2 30 ± 5.8 pO2 (mmHg) 150 ± 10 165 ± 20 14 ± 5 447 ± 50 437 ± 50 213 ± 43 213 ± 43 Hct (%) 45 ± 2.5 47.6 ± 2.3 55 ± 3.3 27 ± 2.5 27 ± 2.5 25 ± 2.8 25 ± 2.8 Lat (mmol/l) 0.7 ± 0.2 1.8 ± 0.7 7.8 ± 2.5 8.7 ± 2.2 8.5 ± 2.4 6.1 ± 1.4 5.1 ± 2 . . . . Slow . Fast . Slow . Fast . . Basal . 18°C . HCA . 45′ CPB . 45′ CPB . 90′ CPB . 90′ CPB . pH 7.39 ± 0.04 7.37 ± 0.04 6.94 ± 0.35 7.4 ± 0.03 7.5 ± 0.03 7.39 ± 0.03 7.38 ± 0.03 pCO2 (mmHg) 37 ± 2.5 34 ± 3 ≥115 23 ± 5.3 21 ± 3.7 31 ± 5.2 30 ± 5.8 pO2 (mmHg) 150 ± 10 165 ± 20 14 ± 5 447 ± 50 437 ± 50 213 ± 43 213 ± 43 Hct (%) 45 ± 2.5 47.6 ± 2.3 55 ± 3.3 27 ± 2.5 27 ± 2.5 25 ± 2.8 25 ± 2.8 Lat (mmol/l) 0.7 ± 0.2 1.8 ± 0.7 7.8 ± 2.5 8.7 ± 2.2 8.5 ± 2.4 6.1 ± 1.4 5.1 ± 2 Non-significant differences in inter-group comparisons were found during the rewarming period (P > 0.05). CPB: cardiopulmonary bypass; HCA: hypothermic circulatory arrest; Hct: haematocrit; Lat: lactate. Open in new tab Inflammation Expression of inflammatory markers in the hippocampal region demonstrated a lower inflammatory response after slow rewarming than after fast rewarming: TNFα (35 ± 9% vs 14 ± 8%; P < 0.01), IL-6 (48 ± 7% vs 19 ± 10%; P < 0.01), CCL5 (35 ± 13% vs 17 ± 15%; P < 0.05) and intercellular adhesion molecule 1 (37 ± 5% vs 26 ± 7%; P < 0.05) (Fig. 2). Figure 2: Open in new tabDownload slide Inflammatory markers. Representative images of the TNFα, IL-6, CCL5 and ICAM-1 expression in the fast (A, D, G and J) and slow (B, E, H and K) groups in the CA1 area of the hippocampal circuit. Graphic representation of the percentage area with inflammatory marker positive staining (C and F: P < 0.01; I and L: P < 0.05). The number of positive marker pixels was normalized to the total number of pixels of the regions of interest considered (magnification ×20). CCL5: chemokine (C-C motif) ligand 5; ICAM-1: intercellular adhesion molecule 1; IL-6: interleukin 6; TNFα: tumour necrosis factor α. Figure 2: Open in new tabDownload slide Inflammatory markers. Representative images of the TNFα, IL-6, CCL5 and ICAM-1 expression in the fast (A, D, G and J) and slow (B, E, H and K) groups in the CA1 area of the hippocampal circuit. Graphic representation of the percentage area with inflammatory marker positive staining (C and F: P < 0.01; I and L: P < 0.05). The number of positive marker pixels was normalized to the total number of pixels of the regions of interest considered (magnification ×20). CCL5: chemokine (C-C motif) ligand 5; ICAM-1: intercellular adhesion molecule 1; IL-6: interleukin 6; TNFα: tumour necrosis factor α. Inflammatory cellular activation in the brain was assessed by microglia activation. Expression of Iba1, a marker of microglia activation, was measured in different areas of the brain: the cerebral cortex, the subventricular zone and the hippocampus, which is particularly susceptible to ischaemic and anoxic damage. In brains subjected to fast rewarming, there was evidence of more significant expression of Iba1 (1.70 ± 0.65% vs 0.94 ± 0.48%; P < 0.05) indicating higher microglia activation (Fig. 3). Figure 3: Open in new tabDownload slide Representative images of Iba1 expression at the cortical level, between the fast rewarming group compared to the group subjected to slow rewarming. In the graph, blind quantification was performed to calculate the average percentage of Iba1 expression in the brain regions considered (**P < 0.01). The number of Iba1 positive pixels was normalized to the total number of pixels of the regions of interest considered (250 images quantified across the different conditions) (magnification ×20). Iba1: ionized calcium-binding adapter molecule 1; TOPRO-3: carbocyanine monomer nucleic acid stain. Figure 3: Open in new tabDownload slide Representative images of Iba1 expression at the cortical level, between the fast rewarming group compared to the group subjected to slow rewarming. In the graph, blind quantification was performed to calculate the average percentage of Iba1 expression in the brain regions considered (**P < 0.01). The number of Iba1 positive pixels was normalized to the total number of pixels of the regions of interest considered (250 images quantified across the different conditions) (magnification ×20). Iba1: ionized calcium-binding adapter molecule 1; TOPRO-3: carbocyanine monomer nucleic acid stain. Oxidative stress Expression of MDA in the hippocampal CA1 region, as a marker of oxidative stress, was significantly higher in the fast compared to the slow group (58 ± 10% vs 26 ± 12%; P < 0.01). This result demonstrates a higher degree of oxidative stress in the fast group (Fig. 4). Figure 4: Open in new tabDownload slide Malondialdehyde expression in fast (A) and slow (B) groups in the anterior and posterior segments of the CA1 area of the hippocampal circuit. Graphic representation of oxidative stress marker (C) (P < 0.01) (magnification ×20). Figure 4: Open in new tabDownload slide Malondialdehyde expression in fast (A) and slow (B) groups in the anterior and posterior segments of the CA1 area of the hippocampal circuit. Graphic representation of oxidative stress marker (C) (P < 0.01) (magnification ×20). Cerebral perfusion and cerebral oedema MRI was used to evaluate cerebral perfusion and cerebral oedema before the experiment and after 2 and after 24 h. The first scan with contrast medium (Gd‐DTPA 300 μmol/kg, Magnevist, Schering, Germany) was performed to assess the degree of cerebral perfusion in the basal ganglia in the fast and slow groups. At 2 h, the values were similar to the basal values for both groups. At 24 h, there was a marked reduction in cerebral perfusion in the fast group compared with the slow group (−6.381E−01 ± 0.01 vs −6.800E−01 ± 0.01). Cerebral perfusion was significantly reduced in the fast group at 24 h compared to the basal values (−6.381E−01 ± 0.01 vs 6.947E−01 ± 0.01; P < 0.05). The mean diffusivity was calculated as an indicator of the degree of cerebral oedema. The analysis showed better values for rats subjected to slow rewarming, with an increase in cerebral oedema in the fast group after 24 h (9.283E−04 ± 0.4E−04 vs 8.488E−04 ± 0.E−04 mm2/s; P < 0.05) (Fig. 5). Figure 5: Open in new tabDownload slide Representative images of mean diffusivity and cerebral perfusion acquisition images (A and B). Comparison of cerebral perfusion and mean diffusivity at 2 and 24 h after the experiment between the 2 groups studied (fast and slow) (C and D). The difference with basal values is statistically significant only after 24 h (*P < 0.05). Figure 5: Open in new tabDownload slide Representative images of mean diffusivity and cerebral perfusion acquisition images (A and B). Comparison of cerebral perfusion and mean diffusivity at 2 and 24 h after the experiment between the 2 groups studied (fast and slow) (C and D). The difference with basal values is statistically significant only after 24 h (*P < 0.05). The same concept was evident after wet/dry brain analysis: there was a tendency towards diminished water content after slow rewarming compared to fast rewarming (Fig. 6). Figure 6: Open in new tabDownload slide Wet/dry ratio of brains in slow and fast groups assessed after surgery (P > 0.05). Figure 6: Open in new tabDownload slide Wet/dry ratio of brains in slow and fast groups assessed after surgery (P > 0.05). DISCUSSION Rewarming is an unconsidered factor compared to cerebral perfusion and hypothermia, but it necessarily follows the others and could influence neurological outcome after HCA. According to current clinical guidelines, the temperature gradient between arterial outflow and venous inflow in the oxygenator during rewarming should not exceed 10°C to avoid outgassing when blood returns to the patient [9]. However, the optimal rewarming protocol is still undefined, as are the pathophysiological mechanisms that play a role in and are influenced by the rewarming rate. In this study, rats were exposed to 60 min of HCA at 19 ± 1°C. The HCA was determined at 60 min in order to induce cerebral ischaemic injury with significant inflammatory reaction, as recently demonstrated by others [15]. Thereafter, 2 different rewarming rates were applied, creating a different temperature gradient between the heat exchanger and the jugular vein temperature probe. In the slow group, we always maintained a gradient of 5°C; in the fast group, we maintained a 10°C gradient until the rats reached 30°C; then we decreased the gradient to 5°C. Compared to slow rewarming, fast rewarming resulted in a significant increase in microglia activation and increased expression of TNFα, IL-6, CCL5 and intercellular adhesion molecule 1 in the brain sections of the cortex and hippocampus. Similarly, Kellerman et al. [16] demonstrated a peak expression of NFkB on the first day after 45 min of HCA. In another study, NFkB levels were demonstrated to be increased in the motor cortex and in the hippocampus, and eosinophilic neurons were significantly higher after fast rewarming compared to slow rewarming [17]. NFkB plays a primary role in regulating the immune response, inflammation, cell proliferation and apoptosis; moreover, its expression is closely correlated with cytokines, receptors and microglia activation assessed in the present study [18]. In support of these results, Berger et al. [19] investigated the inflammatory response to a local ischaemic insult caused by carotid artery occlusion by dividing rats into 3 groups with normothermia, hypothermia with fast rewarming and hypothermia with slow rewarming. The brains were collected after 5 days and showed a decrease in myeloperoxidase positivity in the slow rewarming group. Myeloperoxidase is released into the circulation by polymorphonuclear leucocytes activated in response to various inflammatory processes. In addition, in the samples subjected to slow rewarming, there were reduced concentrations of glutamate involved in the cytotoxic processes, although the results were not statistically significant. An inflammatory response after HCA is crucial in determining neurological outcome. It is responsible for neurocognitive impairment, delirium and temporary neurological dysfunction [5, 6]. However, it is not considered in the clinical setting because it is not possible to measure it using standard diagnostic methods. In this study, MDA, the final product of the polyunsaturated fatty acid oxidative process, was significantly increased after fast rewarming, revealing an amplified oxidative stress compared to slow rewarming. The increase in oxidative stress is easily attributable to the increased inflammatory response that determines a higher production of reactive oxygen species. The development of cerebral oedema was assessed immediately after the experiment by the wet/dry ratio of whole brains extracted immediately after the experiment and after 2 and 24 h by MRI. No statistically significant differences emerged between the 2 groups in the examinations executed after the end of surgery. However, MRI after 24 h demonstrated a significant increase in tissue water content compared to basal values. The accumulation of cerebral fluid has been calculated using water mean diffusivity. The latter is closely correlated with the expression of aquaporin 4, one of the main factors in the formation and resolution of the oedema, and is correlated with brain injury in astrocytes and microglia [20]. Berger et al. [19] found decreased expression of aquaporin 4 in the cortical and subcortical ischaemic areas of animals that underwent slow rewarming compared to fast rewarming. Indeed, in the development of vasogenic oedema, a pivotal role is recognized for several inflammatory mediators that act on the permeability of the blood–brain barrier. Among these are TNFα and IL-6, the expression of which reaches its apex after 24 h [21]. Their expression was significantly increased in the fast group in our study, even immediately after the end of reperfusion. MRI with contrast medium was used to evaluate changes in cerebral perfusion. Only slow rewarming resulted in the recovery of cerebral perfusion. This result might determine improved neurological outcome. Multiple mechanisms are implicated in the development of delayed hypoperfusion, including damage at the level of the endothelial cells, an imbalance in local vasodilator and vasoconstrictors and impaired autoregulation in the setting of decreased blood pressure and temperature [22]. In animal models of cardio-circulatory arrest, interventions aimed at preservation of cerebral blood flow improved neurological outcome [23, 24]. On the other hand, fast rewarming may affect cerebrovascular reactivity and cause hyperaemia [25]. Hyperaemia is detrimental because it intensifies the reperfusion syndrome. Thalamic areas, where early hyperaemia is observed after paediatric cardiac arrest, is characterized by extensive degeneration of neurites and activation of microglia, suggesting an association between early hyperaemia and neurodegeneration [26]. Further investigations need to be carried out in order to establish a universal protocol to ameliorate the results in terms of survival and neurological outcomes. The present study highlights the importance of the rewarming phase after HCA in determining neurological outcome. Further investigations are needed to define survival and neurocognitive outcomes with the perspective of defining the optimal rewarming protocol. Limitations This experimental model was established to study cerebral outcome after HCA and different rewarming rates. Healthy animals without any comorbidities or heart disease were investigated. All risk factors, drugs and surgical procedures related to the clinical setting could theoretically influence the results. In the absence of an extended survival and behavioural analysis, it is not possible to establish neurological outcome and how the different expressions of the detected markers could result in permanent or temporary neurological sequelae. CONCLUSIONS Slow rewarming after HCA might be superior compared to fast rewarming in neurological outcome. The present experimental study demonstrated a reduction in the inflammatory response and in inflammatory cell activation in the brain, reduction in oxidative stress, recovery of cerebral blood flow and reduction of cerebral oedema when a slow rewarming protocol was applied. SUPPLEMENTARY MATERIAL Supplementary material is available at EJCTS online. Funding This study was not supported by any funds but was sustained with internal funds from the Department of Cardiac Surgery of Verona. Conflict of interest: none declared. Author contributions Daniele Linardi: Conceptualization; Investigation; Methodology; Writing—original draft. Beat Walpoth: Conceptualization; Project administration. Romel Mani: Investigation; Methodology. Angela Murari: Investigation; Methodology. Maddalena Tessari: Investigation; Methodology. Stiljan Hoxha: Investigation. Marco Anderloni: Investigation. 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TI - Slow versus fast rewarming after hypothermic circulatory arrest: effects on neuroinflammation and cerebral oedema
JF - European Journal of Cardio-Thoracic Surgery
DO - 10.1093/ejcts/ezaa143
DA - 2020-10-01
UR - https://www.deepdyve.com/lp/oxford-university-press/slow-versus-fast-rewarming-after-hypothermic-circulatory-arrest-FYsPyuwrul
SP - 792
EP - 800
VL - 58
IS - 4
DP - DeepDyve
ER -