Acute cerebral tissue oxygenation changes following experimental subarachnoid hemorrhage

Acute cerebral tissue oxygenation changes following experimental subarachnoid hemorrhage

Critchley, Giles R

Primary brain ischemia following subarachnoid hemorrhage is a major cause of morbidity and mortality. This study aims to determine whether changes in cerebral tissue oxygenation are related to cerebral blood flow changes in the acute phase following experimental subarachnoid hemorrhage. The endovascular puncture model was used to study subarachnoid hemorrhage in male Wistar rats with a tissue oxygenation probe and a laser Doppler probe placed contralateral to the side of hemorrhage. Following the subarachnoid hemorrhage intracranial pressure rose to 53.0 + or – 9.8mmHg (mean + or – SEM). This was associated with a fall in cerebral blood flow to 43.9% + or – 7.1% of its baseline value and a fall in tissue oxygenation to 42.8% + or – 7.7% of baseline. The time course of the fall and recovery in tissue oxygenation was closely correlated to that of the cerebral blood flow (r= 0.66, p = 0.02). The fall in cerebral blood flow was associated with a 42.1% + or – 6.47% fall in the concentration of moving blood cells and a rise of 181.2% + or – 27.2% in velocity indicating acute microcirculatory vasoconstriction. Interstitial tissue oxygenation changes mirrored changes in cerebral blood flow indicating that a change in oxygen delivery was occurring. [Neurol Res 2003; 25: 451-456]

Keywords: Subarachnoid hemorrhage; tissue oxygenation; cerebral blood flow; microcirculatory; vasoconstriction


Cerebral ischemia is a major cause of morbidity and mortality following subarachnoid hemorrhage1,2. Cerebral ischemia may occur immediately following the subarachnoid hemorrhage which may be termed primary cerebral ischemia. Secondary delayed cerebral ischemia or cerebral vasospasm may occur after a delay usually becoming clinically evident between the fourth and 10th day following the hemorrhage. Historically most research has been directed to characterising and treating this delayed cerebral ischemia, with emphasis on changes occurring within the medium sized cerebral vessels of the circle of Willis.

Cerebral blood flow (CBF) changes in the acute immediate phase following subarachnoid hemorrhage (SAH) have been examined in experimental models using laser Doppler flow techniques and hydrogen electrode washout techniques and show an immediate fall in CBF associated with variable recovery3,4.

The development of Clark type polarographic tissue oxygen probes allows continuous, quantitative determination of the partial pressure of oxygen dissolved in the interstitial tissue and hence a measure of the availability of oxygen for cell metabolism5. This is the p^sub ti^O^sub 2^ value or TiO^sub 2^ and is expressed in mmHg as the partial pressure of oxygen. This value represents the balance between oxygen supply and oxygen demand at the cellular level.

Laser Doppler flowmetry provides a continuous local blood flow measurement within a volume of 1 mm^sup 3^ of tissue. The rapid response of the laser Doppler flow signal to changes in local cerebral blood flow (CBF) allows the study of the microvascular hemodynamic parameters following subarachnoid hemorrhage 6. The CBF value may be fractionated into two constituents. The velocity of the cells and the concentration of moving blood cells (CMBC), which provides a measurement of the number of cells within the volume7. Spontaneous rhythmic variations (vasomotion) are seen during the recording of the blood flow. The development of cerebral ischemia has been related to CBF threshold levels in both experimental models8 and in clinical studies9.

The primary aim of this study was to determine whether there was a relationship between the changes in laser Doppler flow and tissue oxygenation following experimental SAH in rats. Changes in the measurements of CMBC and velocity of moving red blood cells and in the frequency of vasomotion were also investigated. Determining the events within the first hours following SAH may give some indication as to the mechanism of the reduction of cerebral blood flow at a microcirculatory level and a potential therapeutic target for improvement in brain oxygenation levels immediately following subarachnoid hemorrhage.


All experimental procedures were performed according to Home Office regulations and in accordance with the Animals (Scientific Procedures) Act 1986 under the appropriate Project and Personal Licences. Male Wistar rats, weighing 330-395 g were used.

A modified endovascular puncture technique3,4 was used to produce subarachnoid hemorrhage in 18 rats, weighing 359 + or – 23 g (mean + or – SD). Seven rats weighing 359 + or – 20 g underwent sham operation and were used as controls.

The animals were anesthetised intraperitoneally with a mixture of fentanyl and fluanisone and midazolam and maintained in deep anesthesia with additional intraperitoneal doses as necessary to prevent reflex contractions to pain. Procedures were performed on a homeothermic operating table to maintain a physiological core temperature of 37[degrees]C throughout the experiment. A tracheostomy was performed, the animal intubated and ventilated with a Harvard rat ventilator to maintain satisfactory arterial blood gases.

A midline dorsal incision was then made over the scalp and the skin and muscles reflected. A right side burr hole was made using a modelling drill behind the coronal suture and 3 mm lateral to the midline and an electrical strain gauge ICP monitor (Codman, Bracknell, UK) was placed subdurally. Two left sided burr holes were then made in a similar position and a tissue oxygen probe (0.47mm diameter probes CC1-R, Licox, Germany) was placed intraparenchymally through the first burr hole. A laser Doppler probe (0.5 mm diameter 780 nm PF4001, Perimed, Sweden) was placed through the second burr hole. The tissue oxygen and laser Doppler probes were therefore placed contralateral to the side of the endovascular puncture to ensure that measurements were not affected by vessel occlusion by the suture or other local factors. Likewise as the tissue oxygen probe was placed intraparenchymally and the laser Doppler probe was placed in contact with the cortex measured changes were not due to direct contact of the probes with blood released at the time of the hemorrhage but due to underlying microvascular changes. The three probes were fixed with bone wax and histoacryl glue. The laser Doppler signal provided four outputs, the CBF, the CMBC, the velocity and backscatter (BS). The maintenance of a stable backscatter value confirmed constancy of laser Doppler signal. The microtrauma following implantation of the tissue oxygen probes initially results in a low reading10,11 and recording from the tissue oxygen probe was therefore performed once a stable baseline value had been reached. An arterial catheter was placed within the femoral artery to allow access for arterial blood sampling and blood pressure recording.

The subarachnoid hemorrhage was produced after dissection of the right carotid bifurcation facilitated by section of the hyoid. The branches of the external carotid artery (occipital, superior thyroid, ascending pharyngeal and lingual) were diathermied and the artery was sectioned and mobilised. This allowed it to be reflected inferiorly to allow passage of the suture. The only extracranial branch of the internal carotid artery, the pterygopalatine artery, was divided. Aneurysm clips were then placed on the internal and common carotid arteries to isolate the carotid bifurcation. The external carotid stump, held by another aneurysm clip, was then punctured to allow passage of a sharpened monofilament suture. A vicryl suture was then tied around the external carotid stump to allow passage of the Prolene suture but prevent reflux of blood. The clips were then removed from the internal carotid artery and the common carotid artery to allow passage of the Prolene suture intracranially. In the rats undergoing creation of a subarachnoid hemorrhage a loss of resistance felt when the suture was passed 30 mm indicated puncture of the Circle of Willis with a simultaneous rise in ICP. In the sham operated control rats the suture was passed to less than 30 mm, no loss of resistance was felt with no rise in ICP seen. In both groups the suture was then withdrawn and the animal observed for a further 2 h. At the end of this time the animal was sacrificed and subarachnoid hemorrhage confirmed.

Throughout the procedure the mean arterial pressure (MABP), intracranial pressure (ICP), cerebral perfusion pressure (CPP) (calculated as MABP-ICP = CPP), LCBF, velocity, CMBC and TiO^sub 2^ were simultaneously recorded using Perisoft software.

One second data sampling was used. Data was analysed at baseline, at maximum deflection immediately after the SAH, and at 30 min, 1 h and 1 h following the SAH. Two minute intervals were analysed at these time points and mean TiO^sub 2^, CBF, CMBC, velocity and CPP were determined. The frequency of vasomotion was determined in cycles per minute (cpm) counted over the 2 min period. Unless specified data are expressed as mean + or – standard deviation (SD).

Statistical analysis was made using paired and independent f-tests at p

To compare CBF and TiO^sub 2^, area under the curve Pearson Rank correlation was used.


Cerebral perfusion pressure and arterial blood gases were maintained at a constant level during the experiments with no significant differences within the two groups or between groups. The blood glucose rose significantly (p = 0.02) in the SAH group at 1 h following SAH, from 6.4 + or – 0.03 mmol l^sup -1^ to 9.5 + or – 1.1 mmol l^sup -1^ but otherwise remained at a constant level (Table 1).

Fifteen rats (83%) survived 2 h following the SAH. Baseline ICP was 2.8 + or – 4.7mmHg and rose to 53.0 + or – 41.4 mmHg following the SAH. It then took 11.1 + or – 9.7 min to return to a plateau level. Changes in CPP mirrored the ICP profile with a mean fall to 41.6% + or – 30.1% of baseline with recovery over a mean of 9.4 + or – 14.6 min. The CBF reached a nadir of 43.9% + or – 27.6% of its baseline value before returning to baseline after 68.7 + or – 40.2 min in those animals that survived.

The baseline TiO^sub 2^ was 29.4 + or – 12.8 mmHg. Following the SAH the TiO^sub 2^ fell to 42.8% + or – 29.7% of baseline before returning to baseline after 63.8 + or – 37.4 min. A recording from a typical experiment is shown in Figure 1 from a survivor and Figure 2 from a nonsurvivor. In sham operated control animals the maximum deflections from the baseline immediately following the sham operation were not significantly different from either the baseline or the operated group. In these sham operated controls the maximum change in CBF was 89.9% + or – 20.4%, and in TiO^sub 2^ was 102.5% + or – 24.3% with no change in ICP.

The fall in CBF was associated with a 42.1% + or – 24.2% fall in CMBC, measured as the maximum deflection from baseline within 15 min of the SAH. There was an associated maximum rise of 181.2% + or – 101.7% in velocity (Table 2). In the sham operated animals the maximum deflections measured in the same time period were 88.5% + or – 11.7% for CMBC and 98.2% + or – 18.5% for velocity, which were not significantly different from the baseline.

Overall the time course of the fall and recovery in CBF was closely correlated to that of TiO^sub 2^ (r = 0.66, p = 0.02, Pearson rank correlation).

The parameters of vasomotion are shown in Table 3. There was no significant difference frequency of vasomotion at half an hour or 1 h from baseline in the two groups.


Following the SAH there was a significant rise in glucose in the operated group which did not occur in the sham operated group. This presumably represents a metabolic response to the stress of SAH and has been variously reported as a clinical indicator of the severity of the SAH and of poor outcome12,13.

The rise in ICP and its return to baseline is reflected in the changes in CPP. The recovery to baseline of CBF and TiO^sub 2^ is significantly longer. This implies that the partial circulatory arrest that occurs at the time of the SAH is not the sole cause of the prolonged reduction in CBF.

The rise in velocity and fall in CMBC that is associated with the fall in CBF immediately after the experimental SAH indicates that there is a reduction in the number of cells present but those remaining have a greater velocity. This implies that acute vasoconstriction is occurring in the microcirculation following SAH and is responsible for the fall in CBF. Parameters of vasomotion remained constant indicating that microcirculatory vasoparalysis is not occurring within the time measured as has been described in other models14. Recent histopathological studies and clinical studies have also indicated that microcirculatory vasoconstriction occurs after SAH both acutely and after a delay15,16.

A variety of mechanisms have been proposed by which acute microcirculatory vasoconstriction could occur. The microcirculation consists of arterioles, capillaries and venules. These vessels have adrenergic innervation in the rat as well as in other mammals and pre-capillary sphincters exist17. This provides a means by which acute microcirculatory vasoconstriction can occur. Endothelial dysfunction with decreased endothelial nitric oxide synthase activity has been demonstrated in vitro. This leads to an attenuated response to endothelium dependent dilator adenosine diphosphate and accentuated constriction to endothelin-1 in isolated cortical arterioles and may also provide a mechanism for microcirculatory vasoconstriction18. Blocking the formation of 20-hydroxyeicosatetraenoic acid (20-HETE) has been shown to ameliorate the immediate fall in the CBF as measured by laser Doppler flowmetry19. Studies of macrocirculatory changes following experimental subarachnoid hemorrhage20 have shown that acute vasoconstriction is present in the internal carotid and anterior cerebral arteries at 60 min following experimental SAH. Our study demonstrates that acute vasoconstriction also occurs in the microcirculation immediately following SAH.

Tissue oxygenation measurements have been used clinically to determine adverse events following SAH21-23. In this study the immediate events within the first 1.5 h following experimental SAH were investigated as this is a time period which is difficult to study clinically. The changes in tissue oxygenation which occurred closely mirrored the changes in CBF. As the tissue oxygenation measures interstitial oxygen availability this represents the product of oxygen delivery to the cells multiplied by the rate of cellular oxygen utilisation. As the fall in TiO^sub 2^ and subsequent recovery is proportional to the fall in CBF this would imply that over the initial 1.5 hours after experimental SAH, oxygen utilisation by cells is stable whilst oxygen delivery is changing. Whilst intraparenchymal placement of the probe results in local microtrauma, reported as a zone of edema of approximately 0.12mm^sup 11^, this zone of damaged cells does not affect oxygen utilisation and acts as though it is transparent for the TiO^sub 2^ measurement due to the rapid diffusion time of oxygen from the surrounding normal tissue. Therefore immediately following experimental SAH the tissue oxygenation probe gives a similar response to the laser Doppler probe measuring CBF.


We have demonstrated that the immediate reduction in CBF that occurs following experimental subarachnoid hemorrhage is due to acute microcirculatory vasoconstriction. Tissue oxygenation changes mirror changes in CBF indicating a change in oxygen delivery at the cellular level. These observations on the immediate microcirculatory response to SAH throw further light on primary cerebral ischemia following SAH.


This study was funded by a grant from the Neurosciences Research Foundation (UK Charity Registration No. 288438). Technical assistance is gratefully acknowledged from Ron Howard of the Medical Physics Department, Atkinson Morley’s Hospital and all the staff at the Biological Research Facilities at St. Georges Hospital, London.


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Giles R. Critchley and B. Anthony Bell

Department of Neurosurgery, Division of Clinical Neurosciences, Atkinson Morley’s Hospital, London, UK

Correspondence and reprint requests to: Giles R. Critchley, Department of Neurosurgery, Hurstwood Park Neurological Centre, Princess Royal Hospital, Lewes Road, Haywards Heath, West Sussex RH16 4EX, UK. [] Accepted for publication March 2003.

Copyright Forefront Publishing Group Jul 2003

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