Studies of isolated global brain ischaemia: II. Controlled reperfusion provides complete neurologic recovery following 30 min of warm ischaemia – the importance of perfusion pressure

Abstract

OBJECTIVES

Neurologic injury after sudden death is likely due to a reperfusion injury following prolonged brain ischaemia, and remains problematic, especially if the cardiac arrest is unwitnessed. This study applies a newly developed isolated model of global brain ischaemia (simulating unwitnessed sudden death) for 30 min to determine if controlled reperfusion permits neurologic recovery.

METHODS

Among the 17 pigs undergoing 30 min of normothermic global brain ischaemia, 6 received uncontrolled reperfusion with regular blood (n = 6), and 11 were reperfused for 20 min with a warm controlled blood reperfusate containing hypocalcaemia, hyper-magnesemia, alkalosis, hyperosmolarty and other constituents that were passed through a white blood cell filter and delivered at flow rates of 350 cc/min (n = 3), 550 cc/min (n = 2) or 750 cc/min (n = 6). Neurologic deficit score (NDS) evaluated brain function (score 0 = normal, 500 = brain death) 24 h post-reperfusion and 2,3,5-triphenyltetrazolium chloride (TTC) staining determined brain infarction.

RESULTS

Regular blood (uncontrolled) reperfusion caused negligible brain O₂ uptake by IN Vivo Optical Spectroscopy (INVOS) (<10–15% O₂ extraction), oxidant damage demonstrated by raised conjugated diene (CD) levels (1.78 ± 0.13 A233 mn), multiple seizures, 1 early death from brain herniation, high NDS (249 ± 39) in survivors, brain oedema (84.4 ± 0.6%) and extensive cerebral infarctions. Conversely, controlled reperfusion restored surface brain oxygen saturation by INVOS to normal (55–70%), but the extent of neurologic recovery was determined by the brain reperfusion pressure. Low pressure reperfusion (independent of flow) produced the same adverse functional, metabolic and anatomic changes that followed uncontrolled reperfusion in seven pigs (three at 350 cc/min, two at 550 and two at 750 cc/min). Conversely, higher reperfusion pressure in four pigs (all at 750 cc/min) resulted in NDS of 0–70* indicating complete (n = 2) or near complete (n = 2) neurological recovery, negligible CDs production (1.29 ± 0.06 A233mn)*, minimal brain oedema (80.6 ± 0.2%)* and no infarction by TTC stain.

CONCLUSIONS

Brain injury can be avoided after 30 min of normothermic cerebral ischaemia if controlled reperfusion pressure is >50 mmHg, but the lower pressure (<50 mmHg) controlled reperfusion that is useful in other organs cannot be transferred to the brain. Moreover, INVOS is a poor guide to the adequacy of cerebral perfusion and the capacity of controlled brain reperfusion to restore neurological recovery. *P < 0.001 versus uncontrolled or low pressure controlled reperfusion

INTRODUCTION

Survival following witnessed cardiac arrest is only 5–15% despite immediate cardiopulmonary resuscitation (CPR), and becomes negligible if CPR is delayed (unwitnessed arrest) or delivered by teams without training [1–3]. Moreover, 33–50% of surviving patients develop significant neurological dysfunction, which occurs in 100% of the rare unwitnessed cardiac arrest survivor [1, 2]. Based upon prior studies of ischaemia and reperfusion in the heart, lung and extremity [1, 4, 5], the most likely primary cause of neurologic damage is an ischaemic/reperfusion (I/R) injury that occurs when normal blood or ‘uncontrolled reperfusion’ is delivered during initial reflow. Reperfusion injury was circumvented experimentally and clinically in the heart, lung and lower extremity by varying the conditions (pressure, flow and temperature) and composition (solution) of reflow to deliver ‘controlled reperfusion’ [1, 4–6], but this biological approach has not yet been investigated for the brain. Nevertheless, we hypothesize that controlled reperfusion is a ‘unifying principle for the biologic process of ischaemia/reperfusion’, and can effectively prevent reperfusion damage in the brain as it did in other organs following prolonged ischaemia.

Brain validation of this concept is essential, since positive findings would reverse the conventional belief that ischaemic brain salvage is impossible following more than 5–10 min of brain ischaemia [1]. Further understanding of this potential requires direct determination of brain recovery following its isolated ischaemia, since limitations follow studies of global sudden death by ventricular fibrillation where cardiopulmonary bypass enhances survival [2]. Such restrictions include (i) secondary brain damage following release of inflammatory mediators from multiple remote ischaemic regions that include the heart, lung and other organs, (ii) generalized inflammation secondary to cardiopulmonary bypass (CPB), (iii) impaired post-resuscitation brain oxygenation and perfusion due to post-operative heart and lung dysfunction which limits cardiac output, ventilation and oxygenation and (iv) inability to completely control all controlled reperfusion facets [1, 2, 4–9]. These limitations are overcome in the regional brain ischaemia model which simultaneously allows the use of the same effective reperfusion system that was employed in prior heart, lung and extremity ischaemia reperfusion studies [1, 4–6]. Consequently, an isolated model allows the remainder of the body to function normally to thereby avoid the aforementioned global problems after sudden death models [1–3, 7].

The recently reported isolated brain ischaemic model sheds light on this process by producing (i) the same global ischaemic injury that follows whole body ischaemia after prolonged ventricular fibrillation [3], (ii) demonstrating that anticipated cerebral flows 550 and 750 cc/min are needed to restore baseline oxygen uptake after transient 30 s ischaemia and (iii) confirming that 450 cc/min reflow was insufficient to return oxygen uptake after only transient ischaemia [3]. The current study employs 30 min of global brain ischaemia in this model to potentially simulate cardiac arrest whereby the victim discovered after 15 min of unwitnessed arrest, and will require the additional 15 min prolongation of ischaemia that is needed to insert femoral–femoral cannulae to initiate cardiopulmonary bypass in the clinical setting of cardiac arrest [1, 10].

Guidelines for proper reperfusion pressure are not known for the brain, and a low reperfusion pressure (30–40 mmHg) has been found to reduce this injury in the heart and lung [4, 5, 9]. However, the aforementioned flows of 550 and 750/min generated reperfusion pressures of >60 mmHg in non-ischaemic animals, which might accentuate brain oedema due to skull restraints, so that lower reflow rates of 350 cc/min were also initially assessed to potentially offset this problem. These lower flow (350 cc/min) studies were also initiated prior to establishing baseline flow characteristics in this model, and actually served as a partial impetus to undertake control flow studies which demonstrated the findings outlined above.

The keynote of this new model analysis is that it may allow formulation of considerations that may be used to initiate emergency CPB in clinical sudden death studies following 30 min of warm brain ischaemia, to thereby allow a 15-min interval for CBP cannula insertion, while avoiding attendant CBP risks. Simultaneously, these isolated brain ischaemia investigations may also mimic regional brain ischaemia after stroke, as well as during circulatory arrest in cardiac surgery, as the ischaemic injury is probably the same in each instance. Controlled reperfusion may find added effectiveness since hypothermia is the only current tool to extend the safe ischaemic interval [11–13].

METHODS

The study protocol was approved by the Institutional Animal Research Committee of the University of California at Los Angeles. All animals received humane care in compliance with the 1996 NRC Guide for the Care and Use of Laboratory Animals.

Seventeen Yorkshire-Duroc pigs (47.2 ± 1.2 kg) were premedicated with 15 mg/kg ketamine and 0.5 mg/kg diazepam intramuscularly. Neuromuscular blockade was achieved by a pancuronium bolus (0.2 mg/kg) that was repeated if needed during the surgical dissection. After endotracheal intubation, anaesthesia was achieved with inhaled isoflurane (1.0–3.0%), and 12–14 breaths/min were provided by a volume-controlled ventilator (Servo 900C, Siemens-Elema, Sweden) at 10–15 ml/kg tidal volume, 4 mmHg positive end-expiratory pressure, with setting adjustment to normalize pH (7.37–7.44) and oxygen (PaO₂ = 80–200 mmHg) and carbon dioxide tension (PaCO₂ = 35–45 mmHg). Anaesthesia was supplemented with fentanyl (1 mg/kg) every hour as needed.

The surgical preparation was previously described [3]. Heart rate and cardiac rhythm were determined by electrocardiography and arterial pressure monitored and arterial blood gases obtained via a left internal thoracic artery catheter. Sterile incisions were performed to expose the needed vessels for cerebral ischaemia. Intravenous heparin (250 IU/kg) was given after all surgical dissections were complete, but before clamping vessels to induce cerebral ischaemia. A Swan-Ganz catheter (Model 132F5, Baxter healthcare Corp., Irvine, CA, USA) was inserted via the right external jugular vein and guided into the pulmonary artery, and a venous catheter was fixed to the bulb of the right internal jugular vein for venous pressure and blood draws to determine cerebral metabolic values and oxygenation. Cefazolin (30 mg/kg, GlaxoSmithKline, Philadelphia, PA, USA) was given every 8 h and saline was infused at 5–10 ml/kg/h. Body temperature was kept at 37°C using a heating pad and lamp as needed. An in vivo optical spectroscopy (INVOS) cerebral oximeter (INVOS model 4100; Somanetics, Troy, MI, USA) patch was placed on the forehead avoiding the sagittal sinus area, and cerebral oxygen saturation recorded continuously.

Experimental protocol

A new model of isolated complete (global) brain ischaemia described previously [3] was used to produce 30 min of global brain ischaemia in each pig. Carotid artery inflow was occluded by clamping the pig's innominate artery which has left and right carotid and right subclavian artery branches. Vertebral artery inflow was prevented by clamping the left subclavian just distal to the aortic arch, both internal mammary arteries and both subclavian arteries just beyond their mammary arteries. Interrupting cerebral blood flow caused a sudden catecholamine surge producing hypertension and tachycardia which was controlled by titrated infusions of nitroprusside and esmolol to maintain normal haemodynamic stability. Hallmarks for complete brain ischaemia included severe hypertension from a sudden catecholamine surge, immediate INVOS (oximeter) reduction to 15% (lowest level measured), dilated non-reactive pupils and carotid pressure <12 mmHg with a jugular venous pressure of <8 mmHg [3]. Following 30 min of isolated warm global brain ischaemia pigs received either uncontrolled or controlled reperfusion as detailed below.

Reperfusion characteristics

Uncontrolled reperfusion (normal blood)

Reperfusion with normal blood was accomplished in six pigs by removing all the vascular clamps.

Controlled reperfusion

‘Controlled reperfusion’ was delivered for a 20-min interval at 37°C with the solution and perfusion method described below, and then all vascular clamps were removed to resume normal blood circulation. The reperfusion system was of the same type that was previously used in heart and lung reflow studies [4–6]. Controlled reperfusion subgroup flow rates were varied as detailed below.

Three pigs received a 350 cc/min flow rate to maintain a pressure of 30–40 mmHg, employed to simulate the low pressure reperfusion status that was found to be superior in other organs [4–6, 9]. Two pigs received a flow of 550 cc/min, and six pigs received a 750 cc/min controlled reperfusion flow rate.

Delivery method

Blood from the femoral artery (14 French cannula) was mixed at 8:1 ratio with a modified crystalloid solution, passed through a white blood cell (WBC) filter, and using a Quest MPS system (Quest Medical, Allen, TX, USA) delivered into both carotid arteries via 10 French cannula inserted into donor pig arterial grafts (Fig. 1A and B) that were anastamosed to each carotid artery. As detailed in our previous study, right vertebral flow is insured by (i) retrograde carotid flow via the innominate vessel to perfuse the right vertebral artery, which joins the left vertebral to form the basilar artery, and (ii) collateral brain flow via the circle of Willis [3].

Figure 1:

(A) Cerebral perfusion system (B) method of controlled cerebral reperfusion (see text for details).

Solution composition

The modified solution (Table 1) closely mirrors the solution used for controlled reperfusion of the heart, lung and lower extremity [4–6, 9], but was slightly modified by the addition of thiopental and edaverone, and exclusion of high glucose, glutamate and aspartate. Supplemental systemic calcium chloride boluses were required to maintain body ionic calcium levels >0.8 mM/l due to hypocalcaemia that resulted from admixture of the citrate-phosphate-dextrose (CPD) within the modified brain reperfusate with whole body blood volume.

Table 1:

Reperfusate solution

Principle	Method	Final concentration
Provide oxygen	Blood	20–30% Hct.
Buffer acidosis	Tham	pH 7.5–7.6
Limit Ca+2	CPD	Ca+2 0.2–0.4 mM/l
	Mg+2	Mg+2 4–6 mg/l
O2 radical scavenger	Edarovone	2 mg/kga
Lower brain excitation	Thiopental	10 mg/kga
Reduce odema	Osmolarity	>360 mOsm
Exclude WBCs	Pall filter	95% removal

CPD: citrate-phosphate-dextrose.

^aTotal delivered dose.

Post-operative care

Thirty minutes following reperfusion, protamine was given to antagonize systemic heparinization, IV cannulas removed and all wounds closed. A small (20 French) tube was placed in the mediastium for drainage. Pigs were extubated after they regained consciousness, developed spontaneous breathing and could maintain normal PaO₂ and PaCO₂ levels. Pigs were monitored for 24 h, or until premature death. Incidence and duration of post-operative seizures were recorded. Seizures lasting more than a few minutes were treated with IV Midazolam (0.1 mg/kg) which was repeated as needed. Furosimide (1 mg/kg) and mannitol (0.25 gm/kg) were given 1–2 h post-operatively to help reduce brain oedema. Euthanasia was accomplished by intravenous pentobarbital and potassium chloride injection at the completion of each study.

Haemodynamic data

Haemodynamic measurements were made before induction of ischaemia and after reperfusion to determine any change in cardiac function. Cardiac output was determined by triplicate injections of 4°C cold saline solution (thermodilution technique) and cardiac index was calculated using standard equations.

Biochemical data

Arterial and internal jugular blood was sampled at baseline and during reperfusion, immediately centrifuged (3000 rpm) for 10 min, and stored at −20°C until biochemical analysis. Conjugated dienes (CD) levels were determined spectrophotometrically to document oxidant mediated-lipid peroxidation, and expressed as the absorbance at a wave length of 233 nm/0.5 ml plasma after chloroform–methanol 2:1 (vol/vol) extraction. Values were expressed either as total amount in the venous (internal jugular) sample, or production rate, which was only calculated during controlled reperfusion where flow rate was known.

CD production = (venous CD level—arterial CD level) × perfusion flow.

Neurologic deficit score

Neurologic assessment was performed at 4 and 24 h after reperfusion using a species-specific behaviour scale [2, 3] that evaluates five general neurologic examination components with a maximal score of 100 in each category: 0 is normal, and 500 indicates brain death as previously described. Two laboratory team members independently assessed neurologic deficit score (NDS) and the mean value was recorded if different scores were reported.

Tissue oedema and analysis of infarction

After euthanasia, the brain was excised, weighted and placed in the freezer for 10 min. Coronal samples 3–5 mm thick were sliced from the frontal lobe, thalamus and hippocampus and sagittal samples obtained from the posterior brain stem and cerebellum. Tissue oedema was determined within the frontal cortex, hippocampus, cerebellum and brain stem by establishing their immediate weight, and then reweighing them following their 24 h placement in an 80°C oven. Tissue oedema reflected as percent tissue water was calculated as Wet weight—Dry weight/wet weight × 100. The remaining cut brain samples were placed in 1% TTC (2,3,5-triphenyltetrazolium chloride) solution at 37°C for 20–30 min to assess for brain infarcts.

Cerebral oxygen saturation

An INVOS cerebral oximeter was applied to continuously monitor cerebral oxygen saturation. It uses near infrared light to evaluate the cerebral cortex immediately below the sensor.

Cerebral oxygen uptake

Transcranial oxygen uptake was determined by the difference between arterial and venous oxygen content during controlled cerebral blood perfusion. Carotid arterial and jugular venous oxygen content were determined from haemoglobin concentration, percent saturation and dissolved oxygen by the following standard formulas:

where cO₂ is the oxygen content, Hb is the haemoglobin, sat. is the saturation, CMRO₂ is the transcranial oxygen consumption, cAO₂ is the arterial oxygen content, cSjO₂ is the internal jugular venous blood oxygen content and CBF is the cerebral blood flow.

Statistical analysis

Data were expressed as mean ± SEM. Two-way analysis of variance with Fisher's least significant difference procedure for post hoc repeated measurements was used to analyse intra- and intergroup differences. Perioperative characteristics of groups were analysed by Student's t-test for continuous data and χ², or by Fisher's exact test for categorical data. P < 0.05 were considered statistically significant.

RESULTS

Baseline and post-reperfusion blood pressure cardiac output, and pulmonary function (blood gas and pulmonary pressures) were similar in pigs receiving uncontrolled or controlled reperfusion. No pig required inotropic support, demonstrated pulmonary comprise or required a blood transfusion.

Sudden interruption of brain blood flow caused a catecholamine surge characterized by hypertension and tachycardia within 60–90 s, and this response required intravenous nitroprusside and esmolol to maintain normal haemodynamic stability. Drug infusion was usually needed for only 15–20 min, with their frequent discontinuation prior to the end of 30 min ischaemia. The INVOS cerebral oximeter consistently abruptly fell to the lowest level of 15%, the pupils became dilated and non-reactive and carotid artery pressure fell to <12 mmHg while simultaneous jugular venous pressure (CVP) was 6–8 mmHg to imply the absence of blood flow.

Uncontrolled reperfusion

Normal blood reperfusion immediately raised internal jugular vein and brain oxygen saturations (INVOS) >85–90% (<10–15% O₂ extraction) in all six pigs (Fig. 2). CD rose to 1.78 ± 0.13 A233 nm/0.5 ml as shown in Fig. 5. The 24 h NDS was 249 ± 39 in the five of six surviving pigs as shown in Table 2. Every pig developed post-reperfusion seizures and one pig succumbed at 14 h after developing hypertension, bradycardia, fixed dilated pupils and then absent respiration. Autopsy following this early death showed marked oedema that is consistent with brain herniation. Post-mortem examination in five survivors demonstrated macroscopic swelling and oedema formation (Table 2) in cortex, cerebellum and hippocampus. Extensive brain infarction was evident by TTC stain (Fig. 6) in each pig.

Figure 2:

Surface brain oxygen saturation measured by INVOS during reperfusion. *P < 0.05 uncontrolled versus controlled (high and low pressure) reperfusion.

Figure 5:

(A) Brain (internal jugular vein) CD (measure of oxygen free radical formation) levels during reperfusion; (B) CD production during controlled reperfusion. Note: High pressure controlled reperfusion not only limited overall release of CD, the production actually decreased during reperfusion. Control high and control low = controlled reperfusion pressure, *P < 0.05 versus control high.

Table 2:

Neuro-deficit score and brain oedema (percent water) post-reperfusion

Neuro-deficit score			Tissue oedema
Reperfusion	4 h	24 h	Cortex (%)	Hippocampus (%)	Cerebellum (%)
Laboratory controls	0	0	81.5 ± 0.2	77.3 ± 0.6	81.1 ± 0.3
Uncontrolled	261 ± 43*	249 ± 39*	84.4 ± 0.6*	81.7 ± 0.9*	84.6 ± 0.6*
Controlled
Low	249 ± 26*	236 ± 22*	85.5 ± 0.3*	81.4 ± 0.8*	83.4 ± 0.5*
High	66 ± 14	25 ± 29	80.6 ± 0.2	77.4 ± 0.4	81.6 ± 0.3

*P < 0.001 versus laboratory controls and high pressure controlled reperfusion; low and high refer to reperfusion pressure.

Figure 6:

TTC stain showing areas of infarction. On the left and middle, a typical example of the TTC stain in a pig undergoing 30 min of ischaemia followed by uncontrolled reperfusion or controlled reperfusion with low pressure. Note the marked oedema collapsing the ventricle and infarctions in the basal ganglia and watershed areas of the cortex. In contrast, on the right a typical section from a pig receiving high pressure controlled reperfusion. Note the lack of oedema with preserved ventricle and no evidence of brain infarction.

Controlled reperfusion

All 11 pigs receiving controlled reperfusion survived 24 h. The initial three pigs received flow at 350 cc/min to maintain a pressure of 30–40 mmHg to match the successful inflow pressures in prior heart and lung studies [1, 4–6, 8, 9]. However, at this flow INVOS surface cerebral oxygen saturations during reperfusion were low (35–40%), and post-reperfusion they developed marked oedema (85.1 ± 0.7%), and severe neurologic damage (NDS 236 ± 22). This led us to perform baseline perfusion studies [3], which showed that even higher flows (450 cc/min) were insufficient to adequately perfuse the brain in this model, and so these three pigs were thereby excluded from subsequent analysis. Reperfusion carotid pressures in two pigs receiving flows of 550 cc/min are shown in Fig. 3, and a similarly low carotid pressure existed in two of six pigs receiving flows of 750 cc/min, due it a low vascular resistance in these pigs despite these higher flow rates. In contrast, carotid pressure was significantly increased in four of six pigs receiving controlled reperfusion at 750 ml/min. Figures 2–6 display overall results during the 20 min of controlled reperfusion, and this separation is based upon carotid perfusion pressure, rather than flow rate.

Figure 3:

Cerebral perfusion pressure during controlled reperfusion. Control high and control low = controlled reperfusion pressure, *P < 0.05 High versus low pressure.

All eight pigs exhibited normal brain oxygen (INVOS) saturations of 50–70%. However, transcranial oxygen uptake during reperfusion (Fig. 4) was reduced in each of the four pigs exhibiting the lower pressure. Post-operative seizures developed in three of four of pigs receiving low pressure reperfusion, their NDS was 236 ± 22, and Table 2 and Figs 5 and 6 document the extent of brain oedema, CD augmentation and brain infarction by TTC stain. Each of these adverse post-reperfusion alterations following controlled reperfusion at lower pressure reflected the same severe modifications that developed following uncontrolled reperfusion with normal blood.

Figure 4:

Global brain oxygen consumption during controlled reperfusion. Control high and control low = controlled reperfusion pressure, *P < 0.05 High versus low pressure.

Conversely, each of the four pigs receiving controlled reperfusion that maintained a higher carotid pressure exhibited elevated brain oxygen uptake, and minimal initial CD production that further declined during the 20 min reperfusion interval. There was the absence of seizures and 24 h NDS ranging between 0 and 70 indicating either complete (n = 2) or near complete (n = 2) neurological recovery whereby each could sit, eat and drink. Cortex, hippocampus and cerebellum oedema formation was minimal (Table 2). No infarction was observed by TTC staining as shown in Fig. 6.

DISCUSSION

These findings demonstrate the first evidence that (i) brain injury can be avoided after 30 min of cerebral ischaemia, a time previously thought to produce irreversible brain damage, provided that controlled reperfusion conditions are varied to deliver a carotid pressure of >50 mmHg, (ii) the low pressure method of delivering controlled brain reperfusion at carotid pressures <50 mmHg is ineffective in restoring neurological recovery, despite its usefulness in other organs [1, 4–6, 9] and (iii) increased global oxygen consumption is predictive of functional recovery, whereas normal INVOS measurements provide a poor guide to improving neurological outcomes. These observations show that the brain's ischaemic state introduces a vulnerability to damage that may either be accelerated by reperfusion with normal blood (uncontrolled reperfusion), or is modified by controlled brain reperfusion in a manner similar to the method that was previously successful in avoiding irreversible heart, lung and lower extremity injury [1, 4–6, 9].

These individual organ studies created the infrastructure towards addressing controlled brain reperfusion. The first step required creation of a model of isolated global brain ischaemia, and identifying changes after ischaemia in this model, before studying whole body sudden death. This isolated brain ischaemia approach mimics prior studies in other individual organs, and simultaneously excludes secondary neurological effects after sudden death, where remote ischaemic organ damage may impair brain recovery through the liberation of post-reperfusion inflammatory mediators or organ dysfunction, and CPB may cause whole body inflammatory changes [1, 3, 5, 6, 14]. The genesis of the current isolated brain ischaemic investigations stemmed from several sources that include (i) prior evaluation following prolonged hypothermic circulatory arrest, where modification of the pump prime with a controlled reperfusate resulted in recovery of individual organs such as the heart, lung, liver and most importantly, the brain [11, 14], (ii) a recent report demonstrating complete neurologic recovery following 15 min of normothermic ischaemia from sudden death by modifying the pump prime [15] and (iii) failure of CBP to achieve neurologic recovery following 15 min of sudden death if immediate CPR was undertaken, as CPR delivered uncontrolled reperfusion with normal blood before the controlled reperfusion was delivered by the modified CBP prime [2, 15]. The two conclusions that arise from these observations include (i) CPR after unwitnessed (prolonged) arrest will accentuate damage by delivering uncontrolled reperfusion, implying that the current clinical therapy (CPR) may need to be abandoned in the setting of unwitnessed arrest if neurologic recovery is to be obtained, and (ii) studies of a longer interval of at least 30 min of global ischaemia are needed in the clinical setting to provide the time frame needed to perform the percutaneous cannulation required to deliver the controlled reperfusate [1, 2, 10, 15].

The importance of delaying treatment following prolonged organ ischaemia until controlled reperfusion may be successfully delivered was shown previously for the heart, where functional myocardial performance was successfully restored after 6 h of coronary ischaemia, an interval that otherwise would lead to severe myocardial infarction [1, 6]. Conversely, myocardial damage was much more severe in both the experimental and clinical setting following delivery of uncontrolled reperfusion with normal blood despite a shorter time interval that was possible by avoiding the requisite delay required to establish CBP and cannulation for controlled reperfusion [1, 6]. Indeed, in every organ(s), we have studied, failure to employ controlled reperfusion after prolonged ischaemia causes extensive metabolic, structural and functional alterations to the ischaemic organ(s), accentuates dysfunction of remote non-ischaemic organs and raises morbidity and mortality [1, 2, 4–6, 9, 11, 14].

The present study confirms that controlled reperfusion may functionally salvage the brain after time intervals previously thought to produce an irreversible injury. The four pig cohort that achieved this positive neurological outcome after receiving higher pressure controlled reperfusion showed that their oxygen free radical production actually declined to normal levels by the end of the reperfusion interval, sustained marginal oedema and their TTC stains demonstrated the absence of brain infarction. This positive effect was only achieved by delivering a reperfusate under the condition and composition that was satisfactory for the brain, and thus differed from conditions in other organs such as the lung, where the absence of lower pressures accentuated damage [1, 4, 5].

Additionally, the effectiveness of the new model of minimally invasive brain inflow occlusion to cause severe global ischaemic isolated brain ischaemia is confirmed as it parallels findings during model development [3]. Each of the six pigs receiving uncontrolled reperfusion demonstrated multiple seizures, oxygen radical damage (high CD), lack of oxygen utilization (high INVOS) during reperfusion indicating damaged mitochondria, high NDS, increased brain oedema and multiple cerebral infarctions. Moreover, lower pressure controlled reperfusion in the four pigs produced the similar functional and anatomic damage observed following uncontrolled reperfusion. These findings substantially differ from how this hypotensive (low pressure) reflow tactic resulted in remarkable outcome efficiency after its application in other organs [1, 4, 5, 9]. Insight into reasons for lower pressure brain controlled reperfusion limitations became evident from observation of infarction by TTC stain that developed in the watershed and deeper cerebral areas, as this observation, thereby implies that perfusion pressure was inadequate to permit its distribution to these areas.

The same anatomic outcome by TTC stain followed both uncontrolled and low pressure controlled reperfusion, but further insight into mechanisms and differences was gained by comparing their reperfusion INVOS recordings. Uncontrolled reperfusion caused extremely high INVOS values, whereas the INVOS levels were significantly lower with low pressure controlled reperfusion (Fig. 2). These findings confirm that low pressure controlled reperfusion salvaged surface cortical brain cells that retained capability to extract oxygen and thus contained intact mitochondria. Conversely, the extremely high INVOS levels following uncontrolled reperfusion imply the presence of mitochondrial damage that likely explains the inability of surface brain cells to utilize oxygen. This implies that controlled reperfusion was effective in regions that received this inflow; but that neurologic injury existed in the watershed and deep infarction regions of the brains of the four low pressure pigs because these areas did not receive the modified solution. These adverse anatomic findings were further exemplified in the three pigs receiving controlled reperfusion at 350 cc/min, where the INVOS levels were quite low (30–40%). Recognition of these findings after 30 min ischaemia in these low flow (350 c/min) pigs was seminal to (i) initiation of prior baseline reflow studies in normal pigs which indentified that even flow rates of 450 cc/min where insufficient in this model to perfuse all areas of the brain [3], and (ii) the 550 and 750/cc/min reflow rates used to determine recovery after prolonged ischaemia in this report.

Neurologic outcomes were closely related to achieving inflow conditions where carotid pressure was at least ∼50 mmHg, a controlled reperfusion condition that existed independent of reflow rates, since ineffective outcomes occurred when lower pressures followed either 550 or 750 cc/min controlled reperfusion delivery rates. Moreover, these adverse prolonged ischaemia observations paralleled baseline findings in non-ischaemic pigs [3] who displayed inadequate brain oxygenation when reflow carotid pressure less than ∼60 mmHg after only 30 s of ischaemia [3]. Specific organ controlled reflow characteristics must be identified for the brain, since lung reflow pressures of 20–30 mmHg were essential to achieve full recovery after 2 h of ischaemia [1, 5, 9], whereas in the brain, these low pressures led to multiple infarcts.

These observations only reflect preliminary findings, since the improvement observed at high pressure controlled reperfusion must be balanced against their potential to create brain tissue oedema following expansion of the reperfused brain within its rigid skull surroundings [4–6, 8, 9]. This issue is further addressed in the subsequent study of 30 min of isolated brain ischaemia [16], which evaluates the role of pulsatile perfusion, since this modality maintains a lower mean pressure while simultaneously assuring a higher peak pressure that may improve controlled reperfusion distribution into deeper vascular beds [17].

One question that must be answered is why the perfusion pressure was lower in two of the animals receiving a flow of 750 cc/min. Possible explanations include drug effects from either isoflurane or residual nitroprusside effects, or an intrinsic vascular abnormality. Among these, isoflurane may have been a critical factor, since it is a vasodilator [18–20], and was (i) delivered at higher concentrations during ischaemia to lower vascular resistance from the catecholamine surge, and (ii) then maintained at that dose in some animals during reperfusion. This effect was tested in separate studies in normal pigs without ischaemia [20], where higher isoflurane concentrations (3%) at fixed flow rates lowered carotid inflow pressures, raised surface brain oxygen levels (INVOS), while decreasing global oxygen consumption implying flow redistribution (or ‘steal’) to the brain surface. This effect was reversed by lowering isoflurane levels to 1% [20]. Isoflurane is also neuro-protective since it decreases brain metabolic rate, so that improved brain protection at higher concentrations might be expected, yet did not occur, and we subsequently found that the adverse flow redistribution problem of high dose isoflurane was independent of its metabolic effects [20].

This potential for isoflurane to influence reflow with controlled reperfusion was subsequently evaluated by studying neurologic findings after fixing isoflurane concentrations at <1% following prolonged ischaemia [16]. Nitroprusside is an unlikely cause because it has a short half life, its delivery was usually discontinued during ischaemia, and was thereby absent during the 20 min period of controlled reperfusion. Lastly, no vascular abnormalities were found following post-euthanasia autopsy. Unfortunately, the inference that higher perfusion pressure alone is responsible for insuring adequate flow distribution would be misleading if this endpoint was achieved by delivery of vasoconstrictor drugs, since these agents might alter regional inflow distribution. Consequently, future studies are needed to determine if pharmacologically raising perfusion pressures will provide the same results as obtained by achieving a higher carotid pressure by flow adjustment.

The modified solution (Table 1) used in this study to avoid neurological damage following 30 min of global brain ischaemia closely mirrors the solution used for controlled reperfusion of the heart, lung and lower extremity, since it integrated several compositional elements such as buffers, calcium, WBC's, magnesium, oxygen radical scavengers, osmolarity, nutrients, pressure, flow, temperature etc [1, 4–6, 8, 9, 14]. The brain reperfusate composition also has substantial differences because (i) glutamate was excluded because of concern for possible neuro-excitatory actions [21, 22], (ii) hyperglycaemia was avoided based upon prior concern that it worsened post-ischaemic brain damage [21, 22], (iii) thiopental enrichment lowered brain energy demands and (iv) endaverone was added because it is an oxygen radical scavenger that reduces brain I/R injury in clinical studies [23]. Subsequent investigations must be done to determine their individual, rather than cumulative effects. A similar analysis is also required as future modifications of the reperfusate composition are added. However, it is important to remember that in addition to the solution composition, controlled reperfusion is only successful when it is delivered under the correct conditions [4–6, 8, 9], as the same modified solution was used in all animals, but only the higher pressure pigs recovered.

An interesting observation was that higher global oxygen extraction, characterized by increased oxygen extraction in the high jugular vein during fixed controlled reflow more successfully predicted neurologic recovery than the INVOS oximeter, which was not helpful despite its routine use to clinically gauge the adequacy of cerebral perfusion [24]. This comparison reflects the limitations of INVOS technology, which only measures oxygen levels a few centimetres directly below the sensors, but does not penetrate to examine the deep brain [24, 25]. In contrast, global oxygen consumption, or the more likely measured higher internal jugular venous saturations may better reflect overall brain uptake that includes the superficial, deep and watershed areas. These findings point out that a normal INVOS value may not always signify that perfusion is adequate, even though a low INVOS value predicts brain under-perfusion. These findings thus provide insight into the limitations of normal INVOS readings in patients undergoing CPB, following deep hypothermic circulatory arrest, and/or carotid surgery.

Broad clinical implications would follow confirmation of these findings because they (i) invalidate inferences that irreversible brain damage follows only a few minutes of ischaemia since complete neurologic recovery followed controlled reperfusion after the 30 min ischaemic time frame thought to cause irreparable damage, (ii) contradicts conventional thinking about neurologic resuscitation following unwitnessed arrest in patients with initially untreated sudden death of at least 15 min and (iii) may initiate novel approaches to management of stroke, since a similar I/R injury is probably present.

In conclusion, these studies document that (i) controlled reperfusion allows the brain to be salvaged after 30 min of warm global ischaemia, (ii) neurologic recovery is closely linked to higher cerebral reperfusion pressures and (iii) oxygen consumption is more predictive of cerebral recovery than INVOS, which is a poor guide to trans-cranial brain perfusion.

Funding

This work was supported by a grant from the National Institutes of Health (R01-HL-71729-04).

Conflict of interest: none declared.