Chloralose

The hypothesis was tested in rats that brain ischemia by an intracerebral hematoma can be ameliorated by fibrinolysis and aspiration of the hematoma. Intraparenchymal blood clots were generated by the injection of 50 (mu)I of autologous blood into the right caudate nucleus in two portions seven minutes apart. Thirty or 120 min later 12 (mu)I recombinant tissue plasminogen activator (rtPA) or 0.9% NaCI were injected and after 30 min the resolved hematoma was aspirated. Six hours later cerebral blood flow (CBF) was determined by ^sup 14^C-iodoantipyrine autoradiography. Tissue volumes of CBF

Keywords: Intracerebral hemorrhage; cerebral blood flow; ischemic brain damage; rat

INTRODUCTION

Treatment of spontaneous intracerebral hemorrhage (ICH) is discussed controversially. Whereas a prospective randomized study1 suggests that surgical treatment does not offer any definite advantage compared to conservative treatment, a positive effect of surgical treatment has been shown in circumscribed subgroups of patients in other studies2. In addition conventional craniotomy for evacuation of intracerebral hematomas is being replaced more and more by minimally invasive surgical methods. Since the introduction of local fibrinolysis and drainage of intracerebral hematomas by Hondo and Matsumoto3 in 1984 this method has been widely used and satisfactory morphological results have been reported3-7. However, it remains open whether or not this therapy is superior to conservative treatment.

In principle surgical evacuation may be of advantage since intracranial and local tissue pressure are reduced and toxins and vasoactive substances originating from breakdown products of blood can be removed. Tissue pressure is increased since intraparenchymal blood clots cause compression of the surrounding brain parenchyma resulting in oligemia and ischemia. In addition to the mechanical compression of the surrounding tissue, ischemia may also be enhanced by the vasospastic properties of breakdown products of blood and toxins8. During longer lasting ischemia the zone surrounding the hematoma may become edematous which may further aggrevate brain damage. Edema can be induced by a variety of factors including cysteinyl-leukotriens9, thrombin10, excitatory amino-acids and free radicals11.

Animal experimental studies of ICH have used a model in which a stereotactically implanted microballoon was inflated for a definite time period. Such an intracerebral space-occupying lesion immediately produces a zone of ischemic brain, which then increases over the next four hours, regardless of whether the lesion persists throughout that period or is present only briefly during the initial phase12-15. This model has its limitations since it only allows the study of mechanical effect of intracerebral hematomas whereas biochemical changes induced by the breakdown products of blood are not covered16.

The simulation of intracerebral hemorrhage by infusion of autologous blood into the basal ganglia is a widely used rat model17 but size and extension of the hematoma were not reproducible because the injected blood ruptures either into the ventricular system or extends to the subarachnoid or subdural space. A recently published modification of the infusion model of intracerebral hemorrhage18 allows the production of highly reproducible intracerebral blood clots well amenable to fibrinolytic aspiration. Moreover it has been demonstrated using this model in combination with MR monitoring that fibrinolysis followed by aspiration results in a significant reduction of the hematoma volume19.

This study was carried out to investigate whether fibrinolysis followed by aspiration of intracerebral hematomas is able to reduce the volume of ischemic brain in a rat model.

MATERIALS AND METHODS

A total of 58 male Sprague-Dawley rats each weighing 304.4 +/- 9.4 g (180-450 g) were used for experiments of fibrinolysis and aspiration of experimental intracerebral hemorrhage. Anesthesia was induced with 1.5% halothane, 70% nitrous oxide, 28.5% oxygen and maintained with intravenous chloralose (5 mg h^sup -1^)/urethane (50 mg h^sup -1^) dissolved in saline. Body temperature was kept at 37 deg C using a heating pad. Femoral arterial and venous lines were established for continuous recording of arterial blood pressure and sampling of blood for blood gas analysis as well as withdrawal of blood for production of the intracerebral hematoma and measurement of cerebral blood flow. The rats were tracheotomized and allowed to breathe spontaneously. Animals were placed in prone position and the head was fixed in a stereotactic frame. Five millimetres and 1 mm anterior to the bregma a burrhole was made, the dura was opened and a 25 gauge polyethylene cannula with mandrin was stereotactically placed into the caudate nucleus to a depth of 5 mm at an angle of 20 degrees. The cannula was then sealed in place with glue.

Intraparenchymal blood clots were produced by injection of 50 (mu)l fresh autologous blood into the right caudate nucleus using a double injection technique as described in detail elsewhere20. Briefly, 15 (mu)l of fresh autologous blood is injected and allowed to clot (preclotting) in order to block the way back along the needle track; after seven minutes the actual hematoma is produced in a second step by injecting the remaining 35 (mu)I.

The animals were assigned to the following groups.

Control group

In 8 animals 50 (mu)l of fresh autologous blood was injected without further procedures.

Treatment groups

Treatment after 30 min: Thirty minutes after production of the intracerebral blood clot by injecting 50 (mu)l fresh arterial autologous blood 12 (mu)l recombinant tissue plasminogen activator (rtPA) (Actilyse(R), Thomae, Biberach, Germany) (15 animals) or 12 (mu)l saline (7 animals) were injected into the intraparenchymal blood clot and another 30 min later the resolved clot was aspirated. This rather high dose was chosen to resolve the clot as fast as possible by single injection.

Treatment after 120 min: 120 min after production of the intracerebral blood clot 12 (mu)l rtPA (10 animals) or 12 (mu)l saline (8 animals) was injected into the intraparenchymal blood clot and again 30 min after the resolved clot was aspirated.

Sham-operated animals

In an additional two animals catheters were placed as described but no autologous blood was injected.

Injection of rtPA/saline

To assess the influence of saline or rtPA on SDH staining as well as on cerebral blood flow no autologous blood but 12 (mu)l of saline or rtPA were injected into the right nucleus caudatus of four animals each.

Blood flow measurement

Six hours after production of the intracerebral hematoma cerebral blood flow (CBF) was determined by ^sup 14^C-iodoantipyrine autoradiography using the methods described by Sakurada et al.21 in all animals. 50 (mu)Ci of 4-iodo[N-methyl-^sup 14^C]antipyrine in 1 ml saline are continuously infused at a progressively increasing infusion rate for a period of 1 min. During the 1 min infusion period 14-20 timed blood samples are collected in drops from the free flowing arterial catheter directly onto filter paper discs of 1.3 cm in diameter that have been placed in small plastic beakers and weighed previously. The samples are weighed and radioactivity is estimated with a liquid scintillation counter after extraction of the radioactive compound with etanol. After the 1-min infusion and sampling period the animal is decapitated, and frozen in 2methylbutane chilled to -40 deg to -50 deg C with dry ice. The frozen brains are coated with chilled embedding medium (Lipshaw, Detroit, Ml, USA) and sectioned into 20 Hm sections at -22 deg C in a cryostat. Every fifth section is autoradiographed along with precalibrated [^sup 14^C]methyl methacrylate standards. Local tissue concentrations of ^sup 14^C are determined from the autoradiographs by densitometric analysis. Local cerebral blood flow (LCBF) is calculated from the local concentration of ^sup 14^C and the time course of the blood iodo[^sup 14^C]antipyrine concentrations, including corrections for the lag and the washout in the arterial catheter. LCBF is measured at selected anatomic loci for each side using an image analysis system (MCID, Imaging Research, Brock University, St. Catharines, Ontario, Canada). On the side of the lesion tissue volumes of CBF

Succinate-dehydrogenase staining

To assess histochemically ischemic brain damage around the intracerebral blood clot 20 (mu)m cryosections were taken every 100 (mu)m. These sections were exposed to a reaction mixture containing nitrobluetetrazolium (NBT) which was composed as described by Riddle et al.22. In normal tissue a blue formation was formed due to the activity of the mitochondrial enzyme succinate dehydrogenase (SDH) which is responsible for energy production of the cell. Ischemic tissue that lacks intact mitochondrias and active SDH remains unstained and can therefore be delineated to normal brain tissue. This approach has been used previously in several studies dealing with focal ischemia after middle cerebral artery occlusion23-25. In the present study, this demarcation line between normal and ischemic tissue which is visible at x25 magnification after SDH staining was used to define the 'lesion volume'. Clot volume as well as lesion volume was measured on SDH stained serial sections which were sampled as described above using an image analysing system (Videoplan, Kontron, Eching, Germany).

Statistics

Statistical comparison was performed using the Student's t-test; intergroup comparisons were made with an unpaired t-test, left vs. right hemisphere comparisons with a paired t-test. Differences were considered significant at p

RESULTS

Physiological variables are shown in Table 1. No significant differences of the measured parameters were found between the different groups. Nine animals died before CBF measurements could be performed 6 h after the hematoma was generated. Of these, seven had been treated with rtPA. Bleeding was observed and was the reason for death in four animals treated with rtPA 30 min and in three animals treated with rtPA 120 min after clot generation. Altogether, bleeding was only observed in the 25 rtPA-treated animals (28%). No bleeding was observed in the control group or the saline-treated group, respectively (p

Treatment after 30 min

Fibrinolysis and aspiration 30 min after production of the intracerebral blood clot resulted in a significant (p

The volume of ischemic brain tissue (CBF

Treatment after 120 min

Congruent with the data obtained from the 30 min group, a significant reduction of clot volume was also found when fibrinolysis and aspiration of the hematoma were performed 120 min after production of the intracerebral hematoma. A clot volume of 37.3 +/- 2.9 (mu)l in the saline-treated group (p

There was no statistically significant difference between rtPA treatment after 30 min and 120 min regarding clot volume, lesion volume calculated from SDH staining and tissue volumes showing a CBF

CBF in specific anatomic loci

CBF was measured in 17 specific anatomic loci. In all groups a significant reduction of CBF as compared to the contralateral side was found in regions located close to the intracerebral blood clot. In regions remote from the hematoma no significant difference between the left and right side was found. As an example the data of the animals treated 30 min after generation of the hematoma are shown in Table 2.

Injection of rtPA/NaCI

To verify the extent of lesion which occurs as a result of the injection of fluid into the nucleus caudatus, experiments were performed in which either saline or rtPA were injected. After injection of 12 (mu)l saline or rtPA only traces of blood could be observed along the needle track (n=7). After injection of 12 (mu)l rtPA an intracerebral hematoma of 94 (mu)l was found in one animal. This animal was excluded from further comparison. The volume of the lesion determined from SDH stained sections was 13.3 +/- 4.6 (mu)l, 12.5 +/- 3.5 (mu)l and 3.0 +/- 1.0 (mu)l in saline, rtPA injected and sham operated animals, respectively. Sham operation as well as injection of saline resulted in negligible ischemic brain volumes (0.01 +/- 0.01 (mu)l and 0.1 +/- 0.06 (mu)l, whereas rtPA injection induced an ischemic brain volume of 0.9+/-0.4 (mu)l. This difference was not statistically significant. On the other hand the tissue volume surrounding the needle track which showed a CBF

DISCUSSION

The main problem in previous experimental rat models of intracerebral hemorrhage has been that size and extension of hematomas were not reproducible12,26. Either the injected blood ruptures into the ventricular system or it extends to the subarachnoid or subdural space. Moreover, the effect of treatment could not be investigated before fibrinolytic substances became available. For this reason simulation of intracerebral hemorrhage and of clot evacuation in experimental studies was done by inflation and deflation of a stereotactically implanted microbal loon12-15,27,28.

Recently a rat model of intracerebral hemorrhage has been introduced in which intracerebral bleeding is induced by injection of bacterial collagenase 9. The collagenase attacks the extracellular matrix around the capillaries thus opening the blood-brain barrier3. Thereby a necrotic lesion is produced that resembles hemorrhagic conversion of an infarction31.

A modification of the infusion model of intracerebral hemorrhage18, as used in the present study, allows the production of highly reproducible intracerebral blood clots well amenable to fibrinolytic aspiration. Using this model in combination with MR monitoring it has been shown that fibrinolysis with 12 (mu)l rtPA and aspiration results in a significant reduction of hematoma volume in a rat model, resembling the clinical situation32. Due to rapid clot formation the infusion model of intracerebral hemorrhage is superior to the collagenase model which produces a confluent area of multiple foci of microscopic hemorrhages not amenable to fibrinolytic aspiration.

In a rabbit as well as in a rat model of intracerebral-intraventricular hematomas, produced by injection of coagulated blood, treatment with urokinase resulted in significant clot lysis, as demonstrated in formalin fixed coronal brain sections. One limitation of these studies was that the major part of the hematoma was intraventricular.

Kaufman et al.35 studied safety of 3,750 and 7,500 units of tissue plasminogen activator in a rat model. They found that rtPA caused no apparent allergic reaction in the rat brain, and rtPA did not lead to excessive bleeding, although they found a 'larger clot' in a rat injected 7,500 units TPA. No data are given on the number of rats injected. The rtPA doses used in our experimental study as well as in clinical investi ations4,6 exceed by far the doses tested by Kaufman et al.35. In the present study the injection of 12 (mu)l rtPA into the caudate nucleus of rats did not cause allergic reactions, however severe intraparenchymal hemorrhage was seen in one out of four animals. rtPA caused a significant compromise of CBF around the injection site as compared to injection of NaCI. This may be due to the solvent of rtPA or the drug itself.

Narayan et al.33 treated intracerebral-intraventricular hematomas in rabbits by injection of 10,000 U Urokinase via a ventricular catheter, and no local or other peripheral bleedings were observed, although a mild stimulation of systemic fibrinolysis was seen.

After production of the hematoma treatment with 12 (mu)l rtPA resulted in a 28% rebleeding rate. This may be due to the lesion caused by the catheter implanted shortly before application of the fibrinolytic drug concomitantly with the high dose of rtPA. This rather high dose was chosen to resolve the clot as fast as possible by a single injection. Aydin et al.34 reported a mortality rate of 32% due to rebleeding by treating a 50 (mu)l clot of injected coagulated blood in rats with 5,500 units of Urokinase (UK) 2 min after production of the hematoma. After readjustment of the dose to 2,200 units UK no rebleeding was observed.

As stated above the microballoon rat model with inflation and deflation of the balloon for a definite time period was used to study the effects of treatment of an intracerebral hematoma in lack of a better animal model. * A

Kingman et al.14 studied the effects of an inert mass lesion, a 50 (mu)l microballoon, on regional cerebral blood flow. In the 'acute' observaton the balloon in one group was expanded for 2.5 min, deflated and CBF was measured 5 min later, compared to CBF measurements 5 min after continuous expansion of the microballoon in another group. In a 'chronic' observation the balloon was inflated for 2 h and CBF was studied 30 min later compared to CBF measurements after continuous expansion of 2.5 h. The time for restitution of cerebral blood flow was 5 min and 30 min, respectively. They found that the focal ischemic lesion caused by a 50 (mu)l microballoon enlarged with time, and that simulated removal of the lesion did not alleviate the volume of perifocal ischemic tissue.

In another study of the Glasgow group13 a microballoon was temporarily expanded for 10 min and CBF was measured 4 h later. It was found that ischemic damage and reduced CBF persisted for 4 h. For comparison a control group without inflation of the balloon was studied, however, a group in which the balloon was continuously kept inflated for 4 h was not investigated. Nevertheless the authors concluded that operative removal of the mass at any stage will limit the eventual lesion size

In a consecutive study by this group15 results from expanding the balloon for 5 min or 4 h supported the assumption that the initial ischemic lesion produced does not remain static, but progresses with time and is significantly larger after 4 h than after 5 min. They concluded that therapy directed at this subsequent progression of ischemia may be beneficial in reducing the ultimate amount of ischemic brain damage.

A subsequent study showed that early removal is able to improve late neurological outcome, while histological findings and brain specific gravity data showed only trends consistent with a less severe effect of transient balloon inflation as compared to a continuing mass lesion28.

All these studies with microballoons, however, only allow the study of the mechanical effect of intracerebral hematomas such as tissue disruption and local or compartmental pressure differences, while not taking into account the fact that biochemical effects might exert considerable influence by provoking secondary effects16.

The present study indicates that injection of 12 (mu)l rtPA results in resolution of the major part of the hematoma and a significant reduction of the volume of ischemic brain with CBF of 10 ml 100 g^sup -1^ min^sup -1^ or less irrespective of the time interval (30 min or 120 min) after production of the hematoma. The tissue volumes with CBF levels up to 30 ml 100 g^sup -1^ min^sup -1^ as well as those with reduced energy metabolism depicted on SDH stained sections showed no difference between treated and untreated animals.

Further studies have to demonstrate whether the tissue of the penumbra-zone is underway to irreversible ischemic damage or to recovery. Another aspect to be addressed in further studies is the role of time interval between stroke and decompression by clot removal, since animals were not treated later than 120 min after generation of the hematoma.

As the hemostatic system of animals may be different from that of humans so the physical and biochemical characteristics or the clots and their susceptibility to lytic drugs may vary greatly from human hematomas , and it remains to be evaluated whether, and if so how far, observations of in vivo and in situ dose effects correlate with reactions of intracerebral blood clots in patients. However, the goal with the present model is primarily to study prognostic changes for the brain.

ACKNOWLEDGEMENTS

This study was supported by a grant (DE 597,1-1 ) from the Deutsche Forschungsgemeinsschaft (DFG) to Dr Wolfgang Deinsberger.

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Wolfgang Deinsberger*, Johannes Vogel^, Casten Fuchs*, Ludwig Michael Auert^^(sec), Wolfgang Kuschinsky^ and Dieter Karsten Boker*

*Neurosurgical Clinic, Justus-Liebig University Giessen

^Department of Physiology, University of Heidelberg

^^Neurosurgical Clinic, University of Saarland, Homburg/Saar

(sec)Institute of Applied Sciences in Medicine, Munich, Germany

Correspondence and reprint requests to: Wolfgang Deinsberger, MD, Neurosurgical Clinic, Justus-Liebig University Giessen, Klinikstr. 29, 35385 Giessen, Germany. Accepted for publication March 1999.

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