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Standardized induction of subarachnoid hemorrhage in mice by intracranial pressure monitoring

May 2010
Journal of Neuroscience Methods 190(2):164-70

May 2010
190(2):164-70

DOI:10.1016/j.jneumeth.2010.05.005

Source
PubMed

Authors:

Benjamin Friedrich

Temedica

Karsten Schöller

Schön Klinik Hamburg

Serge C Thal

Universität Witten/Herdecke

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Subarachnoid hemorrhage (SAH) is the subtype of stroke with the most unfavorable outcome but the least well investigated molecular pathophysiology. Among others, not sufficiently well standardized in vivo models suitable for the use with transgenic animals may be responsible for this situation. Therefore the aim of the current study was to detect suitable intra-operative parameters for the controlled and standardized induction of SAH in mice and to characterize the long-term functional and histopathological outcome of mice subjected to this procedure. Experimental study in mice using the intraluminal Circle of Willis perforation (CWp) model of SAH. SAH induced a sharp increase of intracranial pressure (ICP) from 5.1+/-1.2 to 78.5+/-9.3 mm Hg (mean+/-SD; p<0.05), a concomitant drop of cerebral blood flow (rCBF) by 81+/-4% (p<0.05), and a significant Cushing reflex response (p<0.05). rCBF measurements alone could not reliably detect SAH. SAH resulted in significant brain edema formation (brain water content increase at 72 h: 2.9+/-0.9%; p<0.05), loss of hippocampal neurons (CA1: -56%, CA2: -55%; CA3: -72%; 7 days; p<0.05), severe neurological dysfunction over 7 days, and a mortality of 30%. Our results indicate that CWp in mice can be standardized by intra-operative ICP monitoring. CWp leads to prolonged intracranial hypertension, selective neuronal cell death in the hippocampus, and severe neurological dysfunction. CWp in mice with ICP monitoring may therefore become a valuable tool for future investigations of the molecular pathophysiology of SAH.

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Standardized induction of subarachnoid hemorrhage in mice by

intracranial pressure monitoring – Characterization of the model

Sergej Feiler*, Benjamin Friedrich*, Karsten Schöller, Serge C. Thal, and Nikolaus Plesnila

Institute for Surgical Research and Department of Neurosurgery, University of Munich

Medical Center - Grosshadern, Marchioninistr. 15, 81377 Munich, Germany

*both authors contributed equally to this work

Address of correspondence:

Prof. Nikolaus Plesnila

Royal College of Surgeons in Ireland (RCSI)

123 St. Stephen’s Green,

Dublin 2, Ireland,

Phone: +353 1 402 2794,

E-mail: nikolausplesnila@rcsi.ie

Feiler et al., Brain damage and ICP after SAH in mice

ABSTRACT

Background and Purpose: Subarachnoid hemorrhage (SAH) is the subtype of stroke with

the most unfavorable outcome but the least well investigated molecular pathophysiology.

Among others, not sufficiently well standardized in vivo models suitable for the use with

transgenic animals may be responsible for this situation. Therefore the aim of the current

study was to detect suitable intra-operative parameters for the controlled and standardized

induction of SAH in mice and to characterize the long-term functional and histopathological

outcome of mice subjected to this procedure.

Methods: Experimental study in mice using the intraluminal Circle of Willis perforation (CWp)

model of SAH.

Results: SAH induced a sharp increase of intracranial pressure (ICP) from 5.1 ± 1.2 to 78.5

± 9.3 mmHg (mean +/- SD; p<0.05), a concomitant drop of cerebral blood flow (rCBF) by 81

± 4% (p<0.05), and a significant Cushing reflex response (p<0.05). rCBF measurements

alone could not reliably detect SAH. SAH resulted in significant brain edema formation (brain

water content increase at 72 h: 2.9 ± 0.9%; p<0.05), loss of hippocampal neurons (CA1: -

56%, CA2: -55%; CA3:-72%; 7 days; p<0.05), severe neurological dysfunction over 7 days,

and a mortality of 30%.

Conclusions: Our results indicate that CWp in mice can be standardized by intra-operative

ICP monitoring. CWp leads to prolonged intracranial hypertension, selective neuronal cell

death in the hippocampus, and severe neurological dysfunction. CWp in mice with ICP

monitoring may therefore become a valuable tool for future investigations of the molecular

pathophysiology of SAH.

Keywords: SAH – mouse – intracranial pressure – neuronal cell death – functional outcome

Feiler et al., Brain damage and ICP after SAH in mice

1. Background

Cerebral hemorrhages cause only 20% of all strokes but are responsible for ~50% of the

entire stroke-related mortality, i.e. hemorrhagic stroke is 4.5 times more fatal than ischemic

stroke (Rosamond, Folsom et al., 1999). One of the most common and severe causes for

hemorrhagic stroke is subarachnoid hemorrhage (SAH), a bleeding from a large extra-

parenchymal cerebral vessel mostly caused by a ruptured congenital aneurysm. SAH has an

over all mortality of about 50% and over 30% of survivors remain severely disabled (van Gijn

and Rinkel, 2001). Despite these facts and some pathophysiological similarities, SAH is by

far less frequently investigated than ischemic stroke. Consequently, several important

aspects of the pathophysiology of SAH, especially on the molecular level, are not well

defined thereby hampering the development of novel therapeutic strategies (Cahill and

Zhang, 2009). One of the reasons responsible for this situation may be that murine models of

SAH which can be used with transgenic animals have so far not been fully characterized and

standardized (Strbian, Durukan et al., 2008). To date important clinical parameters such as

intracranial pressure and neuropathology have not been investigated following experimental

SAH in mice. In contrast to other well standardized murine models of acute brain injury, e.g.

ischemic stroke, successful induction and the severity of the insult are not monitored intra-

operatively leading to heterogeneous results which can only be compensated with group

sizes not easily compatible with difficult to generate transgenic mouse strains and by grading

the severity of experimental SAH only post-hoc (Gao, Wang et al., 2006;Sozen, Tsuchiyama

et al., 2009). This makes a randomized study design difficult to achieve and limits the value

of these otherwise clinically highly relevant animal models significantly (Strbian, Durukan et

al., 2008).

So far several research groups studied SAH in mice either by perforating the Circle of Willis

at the skull base using an intraluminal filament inserted through the external carotid artery

(Kamii, Kato et al., 1999;Sozen, Tsuchiyama et al., 2009;Ishikawa, Kusaka et al., 2009;Liu,

Tang et al., 2007;McGirt, Parra et al., 2002;Parra, McGirt et al., 2002;Saito, Kamii et al.,

Feiler et al., Brain damage and ICP after SAH in mice

2001;Gao, Wang et al., 2006;McGirt, Lynch et al., 2002), by directly injecting blood into the

cisterna magna (Lin, Calisaneller et al., 2003), or by perforating an intracisternal vein (Altay,

Smithason et al., 2009). Since perforation of an intracerebral vessel by a filament resembles

aneurysmal hemorrhage closer than intraventricular injection of blood, Circulus of Willis

perforation (CWp) became the most widely used SAH model in mice. Pioneering studies

using CWp demonstrated delayed cerebral vasospasm (Gao, Wang et al., 2006;Kamii, Kato

et al., 1999;McGirt, Parra et al., 2002;McGirt, Lynch et al., 2002;Parra, McGirt et al.,

2002;Saito, Kamii et al., 2001), neurological dysfunction (Gao, Wang et al., 2006;McGirt,

Parra et al., 2002;McGirt, Lynch et al., 2002;Parra, McGirt et al., 2002;Liu, Tang et al.,

2007;Sozen, Tsuchiyama et al., 2009), brain edema formation (Liu, Tang et al., 2007;Sozen,

Tsuchiyama et al., 2009), and a clinically relevant mortality of ~30% (Gao, Wang et al.,

2006;Liu, Tang et al., 2007;Sozen, Tsuchiyama et al., 2009). So far, however, it remains

unclear how the initial bleeding is linked to these rather late and severe pathophysiological

findings since it is not known if experimental SAH in mice causes intracranial hypertension,

reduces cerebral perfusion pressure, and causes subsequent ischemic brain damage.

Therefore, the aim of the current study was to characterize intracranial pressure (ICP),

cerebral perfusion pressure (CPP), cerebral blood flow (CBF), and neuronal cell death

following experimental SAH and to identify intra-operative monitoring parameters (ICP, CBF)

which may help to further standardize Circle of Willis perforation in mice.

Feiler et al., Brain damage and ICP after SAH in mice

2. Materials and methods

Male C57Bl6 mice (23 to 25 g body weight; Charles River Laboratory, Sulzfeld, Germany)

were used for experiments according to the guidelines of the Animal Care Committee of the

District Government of Upper Bavaria (Protocol Number Az 55.2-1-54-2531-118-05).

2.1. Animal preparation and monitoring

Animals had free access to food and water prior to and after surgery. Anesthesia was

induced by intraperitoneal injection of midazolam (5 mg/kg; Ratiopharm, Ulm, Germany),

fentanyl (0.05 mg/kg; CuraMed, Karlsruhe, Germany), and medetomidin (0.5 mg/kg; Pfizer,

Germany) and maintained by hourly injections of one quarter of the initial dose as previously

described (Thal and Plesnila, 2007). Mice were intubated and mechanically ventilated with

35-38% O2 in room air (Minivent, Hugo Sachs, Hugstetten, Germany). End-tidal pCO2 was

measured on-line with a microcapnometer (CI240, Columbus Instruments, Columbus, USA).

A thermostatically regulated, feedback-controlled heating pad (FHC, Bowdoinham, USA) was

used to maintain rectal temperature at 37°C. Anesthesia was terminated by intraperitoneal

injection of atipamezol (2.5 mg/kg; Pfizer, Karlsruhe, Germany), naloxon (1.2 mg/kg; Inresa,

Freiburg, Germany), and flumazenil (0.5 mg/kg; Hoffmann-La-Roche, Grenzach-Wyhlen,

Germany). To prevent hypothermia animals were kept in an incubator at 33°C ambient

temperature for 24 hours after surgery.

2.2. ICP measurement and laser-Doppler flowmetry

ICP was continuously measured in the epidural space of the right hemisphere from 40

minutes before until 30 minutes after SAH using a Codman ICP microsensor (Johnson &

Johnson Medical Limited, Berkshire, UK). To assess ICP 24 and 72 hours after SAH animals

were re-anesthetized, intubated, and the ICP probe was placed for 10 minutes into the

epidural space as described above.

For continuous monitoring of regional cerebral blood flow (rCBF) a flexible laser-Doppler

probe (Periflux 4001 Master, Perimed, Stockholm, Sweden) was glued onto the skull above

the territory of the left middle cerebral artery (MCA). rCBF was measured at a sampling rate

Feiler et al., Brain damage and ICP after SAH in mice

of 10 Hz from 30 minutes before until 30 minutes after SAH. The LDF probe was removed at

the end of surgery.

2.3. Induction of SAH by endovascular filament perforation

SAH was induced by a modification of the endovascular filament perforation method that has

been described in rats and mice previously (Bederson, Germano et al., 1995;Kamii, Kato et

al., 1999;Parra, McGirt et al., 2002). Briefly, animals were anesthetized and placed in a

supine position. The neck was opened by a midline incision and the left carotid artery was

exposed. A 5-0 monofilament was advanced via the external carotid artery (ECA) into the

internal carotid artery (ICA) until the ipsilateral rCBF decreased to ensure the position of the

tip of the filament near the bifurcation of the ICA and the MCA. Then the filament was pushed

further until a sharp increase in ICP indicated the successful induction of SAH. Subsequently,

the suture was withdrawn into the ECA to allow full perfusion of the ICA. In sham operated

animals the same procedure was performed, i.e. the suture was introduced in the ICA,

without causing a rCBF decrease and SAH.

2.4. Neuroscore and body weight

Neurological deficits were assessed daily for 7 days by an investigator blinded towards the

treatment of the animals. Neurological deficits were quantified using a neuroscore as

previously described (Lehmberg, Beck et al., 2000) (Tab. 2). Mice with no neurological

deficits received a score of 0 points, animals with the most severe loss of neurological

function a score of 33 points. Body weight, a sensitive indicator for the overall well-being of

rodents, was assessed daily for 7 days using a balance with movement artifact correction

(Mettler-Toledo, Giessen, Germany).

2.5. Quantification of Cerebral Edema

Animals were sacrificed 24 or 72 hours after SAH and the wet weight (WW) of both

hemispheres was assessed. Thereafter, the hemispheres were dried for 24 h at 110°C and

their dry weight (DW) was determined. Hemispheric water content (%) was calculated using

the following formula ((WW-DW) / WW) x 100.

Feiler et al., Brain damage and ICP after SAH in mice

2.6. Quantification of Ischemic Brain damage

Animals were anesthetized and transcardially perfused with 2% paraformaldehyde (PFA) 7

days after SAH. Brains were removed, dehydrated, and embedded in paraffin. Coronal

sections (bregma -1.6 mm) were prepared and stained with Cresyl Violet. The number of

surviving neurons in the hippocampus was quantified in a region of interest of 0.4 x 0.4 mm

as previously described (Bermueller, Thal et al., 2006).

2.7. Experimental groups

In a first experimental series physiological parameters were assessed and the neurological

function was assessed for 7 days after SAH or sham surgery (n=6 each). In a parallel series

mortality was quantified in SAH and sham operated mice (n=10 per group). In a second

series ICP was measured 24 or 72 hours after SAH or in sham operated animals (n=6 each).

In a third series brain water content was measured 24 or 72 hours after SAH or in sham

operated animals (n=6 each). In a fourth series ischemic neuronal damage was assessed in

sham operated animals or 7 days after SAH (n=6 each). Accordingly, 80 animals were

included in the current analysis.

Animals that died during and after the operation were replaced until the final group size was

n=6. In the series where mortality was assessed animals were not replaced.

2.8. Statistical Analysis

Data are presented as means ± SEM if not indicated otherwise. Statistical analysis was

performed with the software package SigmaStat 3.1 (SPSS Science Inc., Chicago, IL, USA).

Data over time were analyzed with Kruskal-Wallis ANOVA on ranks. The Student-Newman-

Keuls Test was used for multiple comparisons. Mortality was compared between groups with

the Survival Log Rank Test. Statistical significance was assumed at p<0.05.

Feiler et al., Brain damage and ICP after SAH in mice

3. RESULTS

3.1. Physiological Parameters

SAH by filament perforation resulted in an immediate increase of ICP to a peak of 78.5 ± 9.3

mmHg (p<0.05 vs. baseline) followed by a plateau that was reached between 5 minutes

(39.6 ± 8.4 mmHg) and 10 minutes (20.0 ± 3.6 mmHg) after SAH. In sham-operated animals,

ICP remained around the physiological level of 5.0 ± 0.5 mmHg for the whole monitoring

period (Fig. 1A). The increase in ICP resulted in an immediate increase of mean arterial

blood pressure (p<0.05 vs. sham), the well known Cushing response (Fig. 1B), in a

significant drop in cerebral perfusion pressure (CPP) down to 25 mmHg (p<0.05 vs. sham;

Fig. 1C), and the subsequent reduction of cerebral blood flow (CBF) to ischemic levels (19 ±

4% of baseline; p<0.05 vs. sham; Fig. 1D). After ICP dropped to values around 20 mmHg

CCP normalized and CBF recovered to normal values of 80 ± 9.8% of baseline. Except the

Cushing response mice subjected to SAH or sham surgery did not differ concerning blood

pressure and blood gases (Tab. 1).

Vessel perforation resulted in a hematoma at the perforation site (Fig. 1E, dotted circle) and

in a significant extravasation of blood into the subarachnoid space of both hemispheres (Fig.

1E) indicating severe SAH. These changes were only observed when advancement of the

filament into the Circle of Willis was accompanied by a sharp increase in ICP. An isolated

drop in CBF, a method proposed to be useful for monitoring SAH induction in mice, did not

necessarily result in SAH as evidenced by simultaneous ICP measurements (Fig. 1F) and

subsequent visual inspection of blood extravasation. Therefore, ICP measurements are

mandatory in order to reliably monitor experimental SAH in mice.

3.2. Mortality

One animal of the filament perforation group died on postoperative day 1 and another 2

animals died on postoperative day 2 (30% mortality). There was no mortality in sham

operated animals (n=10 each; Fig. 2A).

Feiler et al., Brain damage and ICP after SAH in mice

3.3. Cerebral Edema

SAH by filament perforation resulted in a significant (p<0.05) and continuous increase of

brain water content in both hemispheres up to 72 hours after SAH (Fig. 2B).

3.4. Chronic ICP-Measurement

Brain edema formation resulted in significant intracranial hypertension 24 hours after SAH

(10.0 ± 0.7 mmHg; p<0.05; Fig. 2C). Three days after SAH ICP returned to normal values.

3.5. Brain Damage

SAH in mice did not result in infarction but in selective, bilateral neuronal loss in the

hippocampus (Fig. 3A). Quantification of the number of viable hippocampal neurons 7 days

after SAH revealed a significant loss (p<0.05) of neurons in all regions of the hippocampus

as compared to sham operated controls. The most severe loss was observed in the

ipsilateral CA3 region (Fig. 3B). No cell death was observed in the cerebral cortex or in the

striatum (Fig. 3C).

3.6. Neurological Evaluation and Body Weight

SAH resulted in severe motor and behavioral deficits (15 out of 33 points) during the first 3

days after SAH. Thereafter mice gradually but incompletely recovered until the end of the

observation period as compared to sham operated animals (p<0.05; Fig. 4A). In addition to

the neurological deficits mice of the SAH group exhibited a significant and long lasting loss of

body weight (- 20%) with only minimal recovery (p<0.001; Fig. 4B).

Feiler et al., Brain damage and ICP after SAH in mice

4. DISCUSSION

In the current study we characterized the pattern of brain damage after filament perforation-

induced SAH in mice and demonstrate that the measurement of intracranial pressure is the

only parameter able to reliably detect the induction of SAH by filament perforation.

So far most animals models of SAH were performed on rats, rabbits, cats, or primates (Barry,

Gogjian et al., 1979;Delgado, Brismar et al., 1985;Dorsch, Branston et al., 1989). Despite

multiple advantages, e.g. availability, size, or available standardized models, these species

have, in contrast to mice, the inherent disadvantage of not being amenable to genetic

manipulation. Therefore several groups developed murine SAH models by directly injecting

blood into the cisterna magna (Lin, Calisaneller et al., 2003), dissecting an intracisternal vein

(Altay, Smithason et al., 2009), or perforating an artery at the skull base by introducing a

filament into the cerebral circulation through an extra-cranial approach (Kamii, Kato et al.,

1999;Sozen, Tsuchiyama et al., 2009;Ishikawa, Kusaka et al., 2009;Liu, Tang et al.,

2007;McGirt, Parra et al., 2002;Parra, McGirt et al., 2002;Saito, Kamii et al., 2001;Gao,

Wang et al., 2006;McGirt, Lynch et al., 2002). The cisterna magna injection model and the

venous dissection model can be performed quickly, are highly reproducible due to the

controlled application of the blood, result in an acute increase of ICP and subsequent

decrease of CBF, and lead the development of brain edema and intracranial hypertension as

also observed in man (Broderick, Brott et al., 1994;Claassen, Carhuapoma et al., 2002).

However, both models lack ruptured vessels and endothelial damage which are important

factors of the pathology of SAH (Sobey and Faraci, 1998). Therefore several groups, in

analogy to a model used in rats, developed a murine model where the Circulus of Willis

(CWp) is perforated by an intraluminal filament introduced through the carotid artery. Due to

the obvious replication of the human pathophysiology this model quickly became the most

frequently and widely used experimental SAH model in mice (Kamii, Kato et al., 1999;Sozen,

Tsuchiyama et al., 2009;Ishikawa, Kusaka et al., 2009;Liu, Tang et al., 2007;McGirt, Parra et

al., 2002;Parra, McGirt et al., 2002;Saito, Kamii et al., 2001;Gao, Wang et al., 2006;McGirt,

Feiler et al., Brain damage and ICP after SAH in mice

Lynch et al., 2002). CWp causes delayed cerebral vasospasm (Gao, Wang et al.,

2006;Kamii, Kato et al., 1999;McGirt, Parra et al., 2002;McGirt, Lynch et al., 2002;Parra,

McGirt et al., 2002;Saito, Kamii et al., 2001), neurological dysfunction (Gao, Wang et al.,

2006;McGirt, Parra et al., 2002;McGirt, Lynch et al., 2002;Parra, McGirt et al., 2002;Liu,

Tang et al., 2007;Sozen, Tsuchiyama et al., 2009), brain edema (Liu, Tang et al.,

2007;Sozen, Tsuchiyama et al., 2009), and a clinically relevant mortality of ~30% (Gao,

Wang et al., 2006;Liu, Tang et al., 2007;Sozen, Tsuchiyama et al., 2009). CWp has however,

also some significant disadvantages. So far clinically and pathophysiologically important

findings linking these late changes to the initial bleeding such as intracranial pressure and

neuronal damage were not investigated and CWp has the inherent problem that the volume

of intracranial blood can neither be controlled nor monitored. Accordingly, so far CWp falls

short fulfilling the first and second criteria of an ideal SAH model proposed by Schwartz et al.

which are 1) reproducible and consistent blood deposition in the subarachnoid space 2)

uniform, controlled degree of hemorrhage 3) mechanism of hemorrhage closely simulating

aneurysmal SAH 4) blood distribution correlating with aneurysmal SAH 5) ease of

performance and 6) reasonable costs (Schwartz, Masago et al., 2000). Our results clearly

show that monitoring cerebral blood flow is not sufficient to detect successful induction of

SAH since already the advancement of the filament into the internal carotid artery may cause

a drop of CBF without an increase of intracranial pressure (Fig. 1F). Accordingly, the

increase of intracranial pressure, which is a direct measure for the space occupying effect of

the nascent intracranial hematoma, is the only reliable parameter necessary to be recorded

in order to correctly detect induction of SAH in mice. Since the ICP increase and the absolute

ICP values following SAH are very uniform and show little scattering, we conclude that also

the amount of extravasated blood is quite uniform in mice. Hence, CWp exerts many, if not

all, criteria claimed to be important for experimental SAH (see above).

So far the pattern of SAH induced brain damage was not known in mice. Our results indicate

that CWp in mice causes mainly damage to the hippocampus while the cortex and the

Feiler et al., Brain damage and ICP after SAH in mice

striatum are not significantly affected. Interestingly, except of the ipsilateral CA3 region which

is most severely damaged, cell death affects both hippocampi equally. This indicates that

either cell death is caused by the initial global increase of ICP and subsequent global

ischemia or that the extravasated blood is distributed in a very uniform fashion within the

subarachnoid space thereby causing acute vasospasm and a subsequent reduction of

cerebral blood flow in both hemispheres. We favor the latter explanation due to the actually

demonstrated quite even distribution of blood in the subarachnoid space (Fig. 1E) and some

recent results in man indicating ICP-independent early ischemia after SAH (Schubert, Seiz et

al., 2009). However, only additional experiments measuring absolute levels of CBF with high

spatial resolution by 14C-iodoantipyrene autoradiography at different time points after CWp

and performing experiments on craniotomized animals which will not display the initial ICP

peak will finally prove this hypothesis.

5. Conclusion

Taken together, the current study characterizes the pattern of neuronal damage following the

intraluminal perforation SAH model in mice and demonstrates that the measurement of

intracranial pressure is the only parameter which reliable detects the induction of SAH. The

results of the current study further characterize and standardize the most widely used model

of murine SAH and help to pave the way for further investigations of the molecular

mechanisms of SAH-induced brain damage by using transgenic animals.

Feiler et al., Brain damage and ICP after SAH in mice

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