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# RODENT STROKE MODEL GUIDELINES FOR PRECLINICAL STROKE TRIALS (1ST EDITION)

Shimin Liu, Gehua Zhen, Bruno P Meloni, Kym Campbell, Richard Winn

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Shimin Liu, Gehua Zhen, Bruno P Meloni, Kym Campbell, Richard Winn. RODENT STROKE MODEL GUIDELINES FOR PRECLINICAL STROKE TRIALS (1ST EDITION). Journal of Experimental Stroke & Translational Medicine, 2009. hal-04358217

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RODENT STROKE MODEL GUIDELINES FOR PRECLINICAL
STROKE TRIALS (1ST EDITION)

Shimin Liu, MD, PhD¹*; Gehua Zhen MD, PhD²; Bruno P. Meloni, PhD³; Kym Campbell, BVMS³; H Richard Winn, MD⁴

1 Department of Neurology, Stroke Center, 4 Department of Neurosurgery, Mount Sinai School of Medicine of NYU, New York, NY, USA

2 Department of Anesthesiology, Johns Hopkins School of Medicine, Baltimore, MD, USA

3 Centre for Neuromuscular and Neurological Disorders, University of Western Australia, Australian Neuromuscular Research Institute, Department of Neurosurgery, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia.

ABSTRACT

Translational stroke research is a challenging task that needs long term team work of the stroke research community. Highly reproducible stroke models with excellent outcome consistency are essential for obtaining useful data from preclinical stroke trials as well as for improving inter-lab comparability. However, our review of literature shows that the infarct variation coefficient of commonly performed stroke models ranges from 5% to 200%. An overall improvement of the commonly used stroke models will further improve the quality for experimental stroke research as well as inter-lab comparability. Many factors play a significant role in causing outcome variation; however, they have not yet been adequately addressed in the Stroke Therapy Academic Industry Roundtable (STAIR) recommendations and the Good Laboratory Practice (GLP). These critical factors include selection of anesthetics, maintenance of animal physiological environment, stroke outcome observation, and model specific factors that affect success rate and variation. The authors have reviewed these major factors that have been reported to influence stroke model outcome, herewith, provide the first edition of stroke model guidelines so to initiate active discussion on this topic. We hope to reach a general agreement among stroke researchers in the near future with its successive updated versions.

**Keywords:** Acute stroke; animal model; neuroprotection; middle cerebral artery occlusion; guidelines; consistency

Shimin Liu, M.D., Ph.D.
Department of Neurology, Mount Sinai School of Medicine NYU.
1468 Madison Avenue, New York, NY 10029
E-mail: shimin.liu@mssm.edu
Phone: (212) 241-2252
Fax: (212) 241-6971

Copyright © 2009 SFES 1939-067X/09

<footnote>* Correspondence should be sent to:</footnote>
TABLE OF CONTENTS

<table><tr><td>ABSTRACT</td><td>2</td></tr><tr><td>ABBREVIATION</td><td>4</td></tr><tr><td>DEFINITION</td><td>5</td></tr><tr><td>1. INTRODUCTION</td><td>5</td></tr><tr><td>2. GENERAL GUIDANCE FOR A PRECLINICAL STROKE TRIAL</td><td>5</td></tr><tr><td>3. REVISITING THE STAIR CRITERIA</td><td>5</td></tr><tr><td>4. STROKE MODEL SELECTION</td><td>6</td></tr><tr><td>TABLE 1. SUMMARY OF STROKE MODEL SELECTION</td><td>7</td></tr><tr><td>TABLE 2. COMPARISON OF THE INTRALUMINAL MODEL AND THE TAMURA MODEL</td><td>8</td></tr><tr><td>5. THE INTRALUMINAL MODEL</td><td>9</td></tr><tr><td>A. THE SELECTION OF MCAO OCCLUDINGERS</td><td>9</td></tr><tr><td>B. OPTIMIZATION OF MCAO OCCLUDINGERS</td><td>10</td></tr><tr><td>C. OPTIMIZATION OF SURGICAL PROCEDURES</td><td>10</td></tr><tr><td>6. THE TAMURA MODEL</td><td>11</td></tr><tr><td>A. ABOUT THE MCA OCCLUDINGER</td><td>11</td></tr><tr><td>B. SURGICAL OPTIMIZATION</td><td>12</td></tr><tr><td>C. PURE CORTICAL INFARCTION VERSUS COMBINED INFARCTION</td><td>12</td></tr><tr><td>D. INFARCT VOLUME VARIATION AND OPTIMAL ISCHEMIA DURATION</td><td>12</td></tr><tr><td>7. INFARCTION VOLUME MEASUREMENT</td><td>13</td></tr><tr><td>A. METHODS FOR INFARCTION VISUALIZATION</td><td>13</td></tr><tr><td>B. DIRECT VISUALIZATION OF INFARCT ON TTC STAINED FRESH BRAIN SECTIONS</td><td>14</td></tr><tr><td>C. DIGITAL METHODS FOR DEFINING THE INFARCTED AREA</td><td>14</td></tr><tr><td>D. CALCULATION OF INFARCT VOLUME</td><td>15</td></tr><tr><td>E. CALCULATION FORMULAE FOR INFARCTION VOLUME</td><td>15</td></tr><tr><td>F. MEASUREMENT OF INFARCTION VOLUME VARIABILITY</td><td>15</td></tr><tr><td>8. FUNCTIONAL EVALUATION</td><td>15</td></tr><tr><td>A. THE TIMING OF FUNCTIONAL EVALUATION</td><td>15</td></tr><tr><td>B. EVALUATION SYSTEMS FOR NEUROLOGICAL FUNCTIONAL DEFICITS</td><td>16</td></tr><tr><td>C. ANALYSES FOR NEUROLOGICAL FUNCTIONAL DEFICITS</td><td>16</td></tr><tr><td>9. ANESTHETICS</td><td>16</td></tr></table>
<table><tr><td>SOP:</td><td>Standard Operational Procedures</td></tr><tr><td>STAIR:</td><td>Stroke Therapy Academic Industry Roundtable</td></tr><tr><td>tCCAO:</td><td>Temporary Common Carotid Artery Occlusion</td></tr><tr><td>TTC:</td><td>2,3,5-Triphenyltetrazolium Chloride</td></tr></table>

## DEFINITION

pSDM: percent of SD to mean

## 1. INTRODUCTION

The Stroke Therapy Academic Industry Roundtable (STAIR) was established in order to address the challenges encountered in finding an effective neuroprotective therapy for acute stroke (Fisher et al. 2007; Stroke Therapy Academic Industry Roundtable 1999). Besides the STAIR's concern for study design, other issues relating to experimental stroke research, especially animal stroke modeling have been raised (Dirnagl 2006; Savitz 2007; van der Worp et al. 2005). This is an important issue, and it is now recommended that any stroke model that aims to achieve scientific and therapeutic value must meet certain requirements. The model must be both highly consistent in inducing injury, performed under conditions to avoid co-founding factors influencing outcomes and widely available to most investigators. With the STAIR guidelines providing an excellent framework for the design of preclinical stroke trials, a detailed guidance for conducting individual experiments using stroke models will further improve model consistency, reliability and inter-lab comparability. A review of literature shows that the infarct variation coefficient of commonly performed stroke models ranges from 5% to 200% (see following paragraphs). Many factors play a significant role in causing outcome variation; however, they are not fully defined in the STAIR guidelines. These critical factors include selection of anesthetics, maintenance of animal physiological environment, stroke outcome observation, and animal species used.

Here we provide the first edition of stroke model guidelines (SMG) so as to initiate an active discussion on this topic, with the ultimate aim of reaching a general agreement among stroke researchers in future up-dated versions. Because rodents are the mostly commonly used animals for preclinical stroke trials, the first version of SMG starts with rodent stroke models. In its successive versions, the contents may expand into stroke models of other species. The SMG starts with a general overview of the design and setup of a typical preclinical stroke trial, followed by more detailed guidelines on the implementation of each step.

## 2. GENERAL GUIDANCE FOR A PRECLINICAL STROKE TRIAL

Because investigators have used different units of measurement for the infarction volume and such data have been expressed either in mean ± SD or mean ± SEM, we adapted pSDM (percent of SD to mean,) as the coefficient for infarction volume variation for the purpose of increasing comparability between studies. The SEM, when used by many of the original investigators, has been converted to SD for the calculation of the pSDM.

Before starting an experimental preclinical stroke trial the following steps are necessary for ensuring a good quality study. Specific guidance for most steps is provided in subsequent paragraphs.

1. Revisit the latest STAIR criteria for achieving an optimized study design

2. Select the most appropriate stroke model for your study

3. Determine stroke model parameters, such as anticipated infarct size and surgical procedure

4. Determine the use of anesthetics

5. Determine the components of the inhaled gas

6. Determine the necessity and settings of intubation and ventilation

7. Set up monitoring for arterial blood pressure, blood gases, blood glucose

8. Set up temperature monitoring and maintenance

9. Set up regional cerebral blood flow monitoring

10. Determine a protocol for post-operational care

11. Determine the method and timing for infarct volume measurement

12. Determine the appropriate tests and timing for the assessment of functional deficits

13. Do a pilot study and adjust the experimental settings for further optimization

14. Assess the compliance of the trial with "Good Laboratory Practice" standards.

## 3. REVISITING THE STAIR CRITERIA

### GUIDANCE

The design of a preclinical stroke trial should start with a revisiting of the latest version of stroke therapy academic industry roundtable (STAIR) criteria. Receiving some useful suggestions with caution from the STAIR criteria may help with improving the study design to some extent although there are debates on some issues that STAIR addressed.

### SUPPORTING DISCUSSION
*The STAIR criteria provides some useful recommendations for improving the design of preclinical stroke trials.*

To date, the STAIR group has met six times discussing and revising their recommendations for preclinical and clinical stroke trials (Fisher 2003; Fisher 2005; Fisher et al. 2009; Fisher et al. 2007; Saver et al. 2009; STAIR Group 1999; STAIR Group 2001). Recommendations provided by the STAIR consortia emphasize the design quality of both experimental and clinical stroke trials. With respect to experimental animal stroke trials, STAIR recommendations have highlighted the need for investigators to consider factors such as species and gender differences, clinical relevance of animal models, dose-response determinations, therapeutic time windows, blood-brain-barrier (BBB) permeability and tissue drug levels, treatment randomization, physiological monitoring, and at least 2 outcome measures covering both acute and long-term endpoints.

*The STAIR criteria should be used with caution because there may be conflicts between its suggested late treatment and therapeutic windows.*

In the STAIR I, the ideal neuroprotective drug trial was described thus: "should demonstrate efficacy in at least 2 species, in at least 2 laboratories that use different models, is effective in both permanent and transient focal ischemia, and improves short-term and long-term histological and functional outcomes, even when administered several hours after the onset of ischemia" (STAIR Group 1999). Keeping the therapeutic window in mind, it may be simply impossible to demonstrate robust neuroprotection when the treatment is delivered too late. The therapeutic window is roughly a few hours in rodent MCA occlusion (MCAO) models. For example, in a 300 g rat, a 2-hour duration of transient MCAO produces a large infarct volume of 400-450 mm³, which is similar in size to the infarct caused by permanent MCAO after 24 hours (Greco et al. 2007; Masaoka et al. 2001). Hence, it is likely that a preclinical stroke trial using a 2 hour transient MCAO model and a late treatment time point (e.g., 6h post-MCAO) (Simard et al. 2009; Yin and Zhang 2005) would have missed the therapeutic window and the opportunity to observe a treatment effect. In this instance, a baseline injury quantification study, performed at different treatment time points (e.g.,., 2, 4, 6 hours post-MCAO) would improve study design and increase the chance of obtaining a positive neuroprotective effect. Some histopathological methods and diffusion-weighted imaging techniques described in the infarct volume measurement section of this guideline can be used for the detection and quantification of baseline injury starting several hours after ischemia.

*The STAIR criteria should be used with caution because there may be conflicts between its suggested observational time and the natural history of stroke evolution.*

As discussed in more detail below, both the infarction evolution and changes in functional deficits have their own natural histories. Measuring infarct volume evolution is time sensitive and methodology dependent (see section on Infarction volume measurement). Assessment of functional recovery is even more complicated, is model dependent, and is time sensitive in relation to the recovery pattern and functional test being used (see section of Functional evaluation). Therefore, it may sound arbitrary to recommend all outcome measures be performed in 1-3 days and in 7-30 days.

*Would the STAIR criteria help with increasing the chance of a true discovery (no, but this was not really its aim)?*

As stated in the STAIR I, the purpose of STAIR is “to propose recommendations for ways to optimally preclinically assess neuroprotective and restorative drugs for acute ischemic stroke”. However, these recommendations were condensed into a few designing principles even with the latest update (Fisher et al. 2009), which has been functioning like an elevated threshold limiting experimental discovery entering into clinical trials. These STAIR criteria may help with improving the design quality of preclinical stroke trials, reducing bias and false positive conclusions (Fisher et al. 2009), but has little to do with increasing the chance of scientific discoveries. It is the research direction that holds the chance of scientific breaking through while the optimized methodologies increase the sensitivity for positive findings. The research directions that hold the continued promise for neuroprotection for ischemic stroke has been discussed in our previous review paper (Liu and Levine 2008). Optimization of preclinical stroke trials is more complicated than that the STAIR has recommended, which is what we need to address in this stroke model guidelines

## 4. STROKE MODEL SELECTION

Various stroke models have been developed to mimic different stroke subtypes or pathological mechanisms and can be generally classified into two categories: focal cerebral ischemia models and global cerebral ischemia models. Global ischemia models mimic the clinical conditions of brain ischemia following cardiac arrest or profound systemic hypotension, focal models represent ischemic stroke, the most common clinical stroke subtype. The most commonly used focal ischemia models are the intraluminal filament model (Koizumi et al. 1986) and the Tamura model (Tamura et al. 1981a). Some additional stroke models involve special mechanisms to induce artery occlusion/ischaemia, such as the thromboembolic, endothelin and photochemical models.

### GUIDANCE
There are mainly two factors that influence the selection of in vivo stroke models for preclinical trials. These are the potential protection mechanism of the neuroprotective candidate and the highest achievable model quality with a particular lab setting.

For examples, if the candidate is predicted to reduce ischemic injury by attenuating cerebral edema after thrombolytic therapy, the thromboembolic model should be used; if the predicted neuroprotection is associated with a particular brain cortex region, the photochemical model will be

preferable because this model is able to produce ischemic injury in an arbitrary geometric shape at any location on the brain surface. If the predicted protection mechanism of a drug candidate is shared by several stroke models, the selection of a preferred model could be determined by the achievable model quality, as judged by success rate and outcome consistency. In most cases, the choice is between the intraluminal model and the Tamura model.

<table><caption>TABLE 1. SUMMARY OF STROKE MODEL SELECTION</caption><thead><tr><td>Stroke model</td><td>Preferred application</td><td>Reference</td></tr></thead><tbody><tr><td>Photochemical model</td><td>Neuroprotection is associated with a particular brain cortex region.<br/>Free radicals and endothelial injury play major roles in lesion development.</td><td>(Chen et al. 2004; De Ryck et al. 1996; De Ryck et al. 1989; De Ryck et al. 2000; Eichenbaum et al. 2002; Futrell et al. 1988; Lozano et al. 2007; Ostrovskaya et al. 1999)</td></tr><tr><td>Autologous clot model</td><td>Clot-related protection mechanisms.</td><td>(Beech et al. 2001; Busch et al. 1997; Harada et al. 2005; Kano et al. 2003; Kano et al. 2005; Kilic et al. 1998; Krueger and Busch 2002; Orset et al. 2007; Romanos et al. 2007; Tejima et al. 2001; Toomey et al. 2002; Vosko et al. 2003)</td></tr><tr><td>Endothelin-1 model</td><td>Transient to semi-permanent focal cerebral ischemia that need outcome measurements of both histology and neurofunctions</td><td>(Biernaskie et al. 2001; Horie et al. 2008; Nikolova et al. 2009; Windle et al. 2006)</td></tr><tr><td>Tamura model</td><td>Permanent focal cerebral ischemia.<br/>Transient focal cerebral ischemia in which functional assessment is not needed.</td><td>(Bederson et al. 1986b; Buchhold et al. 2007; Chen et al. 1986; Roof et al. 2001)</td></tr><tr><td>Intraluminal model</td><td>Permanent and transient focal cerebral ischemia that need outcome measurements of both histology and neurofunctions</td><td>(Candelario-Jalil et al. 2008; Khan et al. 2006; Liu et al. 2006; Shimamura et al. 2006a; Shimamura et al. 2006b; Solaroglu et al. 2006; Tsubokawa et al. 2007; Tsubokawa et al. 2006a; Tsubokawa et al. 2006b)</td></tr></tbody></table>

SUPPORTING DISCUSSION

About the photochemical model
Implementation of the photochemical model involves injection of a photosensitive dye that penetrates the BBB. The photochemical reaction produces singlet oxygen and free radicals, which causes endothelial injury and formation of microthromboses. The light used for inducing this reaction can be laser or filtered non-laser light, and can be shone onto a section of artery wall or any location of the skull. Therefore, this model is useful for neuroprotection that is associated with a particular brain cortex region and involves free radical scavenging as a protective mechanism (Chen et al. 2004; De Ryck et al. 1996; De Ryck et al. 1989; De Ryck et al. 2000; Eichenbaum et al. 2002; Futrell et al. 1988; Lozano et al. 2007; Ostrovskaya et al. 1999).

### About the autologous clot model

Although the autologous clot model that mimics thromboembolic stroke has been developed (Kudo et al. 1982), and efforts have been made to improve its outcome consistency, (Wang et al. 2001; Zhang et al. 1997b) this model is still not suitable for validating neuroprotective effects because of its uncontrollable reperfusion and unacceptable variation of infarct area (Wang et al. 2001; Zhang et al. 1997a; Zhang et al. 1997b). Therefore, this model is re-

served for clot-related protection mechanisms which other stroke models cannot address.

### About the endothelin-1 model

Endothelin-1 (ET-1) is a potent vasoconstrictor. It reduces regional cerebral blood flow and produces ischemic injury when being injected directly into brain tissue (Windle et al. 2006) or adjacent to the MCA (Biernaskie et al. 2001; Nikolova et al. 2009). The magnitude and duration of reduction of cerebral blood flow is variable, dose dependent (Nikolova et al. 2009), and strain dependent (Horie et al. 2008), persistent up to 7-16 hours (Biernaskie et al. 2001). ET-1 has a much less potent effect for producing an infarct in mice than in rats (Horie et al. 2008).

### Intraluminal model versus Tamura model.

In experienced hands, the intraluminal model and the Tamura model can achieve similar success rates and outcome consistency (Table 2). However, some types of the Tamura model may cause just cortical injury with small infarction volume, which does not produce consistent functional deficits (Chen et al. 1986; Roof et al. 2001).

<table><caption>TABLE 2. COMPARISON OF THE INTRALUMINAL MODEL AND THE TAMURA MODEL</caption><thead><tr><td>Model</td><td>Intraluminal</td><td>Tamura</td></tr></thead><tbody><tr><td>Need for craniotomy</td><td>No</td><td>Yes</td></tr><tr><td>Incur brain trauma</td><td>No</td><td>Yes</td></tr><tr><td>Occlusion methods</td><td>Monofilaments:<br>heat/flame blunted<br>PLL coated<br>Silicone-rubber coated</td><td>Microclips<br>Suture tying<br>Suture/wire retraction<br>Electrocoagulation</td></tr><tr><td>Success rate for transient MCAO</td><td>60-100%</td><td>40-75%</td></tr><tr><td>Infarct distribution</td><td>Basal ganglia and cortex</td><td>Depending on occlusion site. Only cortical infarct occurs if occlusion site is lateral to the olfactory tract</td></tr><tr><td>Reported best pSDM</td><td>10-20% in rats<br>5-10% in mice</td><td>6.6-16% in rats<br>10-13% in mice</td></tr><tr><td>Functional deficit</td><td>Severe and consistent</td><td>Mild and inconsistent, especially when the infarct is limited to the cortex</td></tr></tbody></table>
For detailed discussion of Tamura model and intraluminal model please refer to the related sections below.

## 5. THE INTRALUMINAL MODEL

For the intraluminal model, the key factors that affect outcome consistency are the **physical properties of the occluder**, the **MCAO surgical procedure** and the strain of animal. Critical physical properties of the occluder that affect stroke outcome include its tip diameter, tip length, tip shape, and flexibility. Some specific surgical procedures have also been developed for different purposes, such as for confirming a successful occlusion, for supplemental occlusion of proximal arteries, and for prevention of premature reperfusion. Animal strain is not the focus of the first version of SMG.

## A. THE SELECTION OF MCAO OCCLUDINGERS

### GUIDANCE

It has been shown that silicone-rubber coated filaments are superior to flame blunted and PLL coated monofilaments for producing consistent ischemic injury (Spratt et al. 2006). There are insufficient data for an accurate comparison between silicon rubber coated monofilaments and glue-coated, resin-coated or nail polish-coated monofilaments. Flame/heat-blunted and PLL coated monofilaments are generally considered unacceptable for neuroprotection studies because of their low success rate, high subarachnoid hemorrhage (SAH) rate, and large variability in infarction volume.

### SUPPORTING DISCUSSION

The intraluminal MCAO models can be induced using different filaments. In the Koizumi model, a silicone-rubber coated monofilament is used, while in the Longa model a flame-blunted monofilament is used. Other occluders include the poly-L-Lysine (PLL) coated monofilament (Spratt et al 2006), methyl methacrylate glue coated monofilament (Shah et al 2006), silicon resin coated monofilament (Yamauchi et al 2005), and nail polish coated monofilament (Matsushima and Hakim 1995). The physical characteristics of the occluder influence outcome variation by causing insufficient occlusion, premature reperfusion, and/or filament dislodgement. The following paragraphs review the MCAO model quality obtained using the most common occluders and their optimizations.

The PLL coated occluders.

The PLL coated monofilament has the lowest success rate, the highest SAH rate and highest mortality rate among all monofilaments in rat models. MCAO models using PLL coated occluders have been reported to have a success rate as low as 13-14% in rats, with model mortality of around 21-31% (Spratt et al 2006). High mortality (50-60%) and infarct size variation have also been reported when using PLL coated sutures in mouse models (Huang et al 1998). While most authors reported low success rates, high SAH rates, and high mortality rates when using PLL coated sutures for both rat models and mouse models, Belayev et al reported increased infarct volume and experimental consistency as compared to uncoated sutures, although in some instances brain infarction did not occur (Belayev et al 1996).

Flame/heat-blunted occluders.

Tsuchiya et al. (Tsuchiya et al 2003) showed that using flame blunted monofilaments to induce MCAO caused a 40% rate of subarachnoid hemorrhage, and pSDM was greater than 100%. In another study (Schmid-Elsaesser et al 1998), models using heat-blunted 3-0 filaments had a success rate of 46% (without further repositioning of the occluder according to laser Doppler flowmetry (LDF) monitoring), with 44% occurrence of SAH. Premature reperfusion occurred very frequently with a rate of 24% when using the heat-blunted filament group as shown through LDF monitoring (Schmid-Elsaesser et al 1998). The authors' own experience confirmed a less than 40% success rate when using flame blunted monofilaments. Personal communication with other MCAO model performers who have experience with the Longa model confirm that a flame blunted occluder is not an acceptable choice for neuroprotection studies. In the mouse intraluminal model, SAH rates can reach as high as 40% if uncoated heat-blunted filaments are being used. In such cases, the pSDM can be more than 50% (Tsuchiya et al 2003).

Silicone-rubber coated occluders.

Studies using silicone rubber coated monofilaments have reported success rates ranging from 66% (Schmid-Elsaesser et al 1998) to 100% (Liu et al 2006), and SAH rates from 0% (Chen et al 2008) to 8% (Schmid-Elsaesser et al 1998). Premature reperfusion rates have been reported to be 26%; readjusting filament location for correcting premature reperfusion could increase the success rate of MCAO (Schmid-Elsaesser et al 1998). The pSDM when using a silicone-rubber coated filament ranges from 30% (Schmid-Elsaesser et al 1998) to around 5% (Maysami et al 2008). It also seems that bilateral laser Doppler flowmetry
try can be a useful tool for detecting premature reperfusion
(Hungerhuber et al 2006).

B. OPTIMIZATION OF MCAO OCCLUDINGERS

GUIDANCE

The physical properties of the occluder tip play a critical
role in causing infarct variation and SAH occurrence. For a
certain range of animal body weights, an optimal occluder
diameter can be found through a series of pilot experi-
ments. It has been reported that the optimal occluder di-
ameter for rats weighing 275-320g is around 0.38 mm for
silicon rubber coated monofilaments (Spratt et al 2006).
The silicone rubber coating length is another important
factor that influences the occluder's ability to block the
back-flow from communicating arteries (Chen et al 2008).
Therefore, an optimal coating length may also exist for an-
imals within a certain body weight range, so matching the
occluder size with animal size would theoretically improve
model consistency. Note too that a shorter coating can
preserve blood supply to the hypothalamus, minimizing
post-surgical thermoregulatory dysfunction, particularly the
occurrence of spontaneous hyperthermia. In order to match
the wide range of rodent animal body weights, a large
number of different occluders in standard size would be
needed. To this end, our recommendation is to obtain
commercially made occluders, which are available in differ-
ent diameters and silicone rubber coating lengths
(www.doccol.com).

SUPPORTING DISCUSSION

When the occluder is matched to animal size, improved success rates and reduced SAH rates can be achieved.

In rat models of 60-min transient MCAO to 24-h permanent MCAO using correct-sized occluders (Candelario-Jalil et al. 2008; Khan et al. 2006; Liu et al. 2006; Shimamura et al. 2006a; Shimamura et al. 2006b; Solaroglu et al. 2006; Tsubokawa et al. 2007; Tsubokawa et al. 2006a; Tsubokawa et al. 2006b), the success rate was found to be 88-100%, and the SAH rate to be 4%. In mouse models of 60-min transient MCAO(Chen et al. 2008; Kleinschnitz et al. 2007; Maysami et al. 2008; Pignataro et al. 2007b; Pignataro et al. 2007c), the success rate was found to be 96% and the SAH rate 0%.

When the occluder is matched to animal size, impressive improvements in infarct consistency have been reported both in rats and in mice.

A technical paper by Shimamura showed consistent infarc-
tion and a tight error bar in rat models even with inexpe-

rienced surgeons (Shimamura et al. 2006a). For a 60-min
to permanent occlusion in rats, the pSDM is around 10% to
20% depending on experimental design and selection of
right-sized filaments. (Candelario-Jalil et al. 2008; Khan et
al. 2006; Liu et al. 2006; Shimamura et al. 2006b; Solaroglu
et al. 2006; Tsubokawa et al. 2007; Tsubokawa et al.
2006a; Tsubokawa et al. 2006b). A 15-min occlusion could
also produce a consistent caudate infarction with little var-
iation in mice (Pignataro et al. 2007a). For a 30-min occlu-
sion, the pSDM was reported to be around 20% in mouse
models (Cho et al. 2007; Kim et al. 2008). Even better con-
sistency has been reported in 60-min MCAO models in
mice, in which the pSDM was around 5-10% (Kleinschnitz
et al. 2007; Maysami et al. 2008; Pignataro et al. 2007b;
Pignataro et al. 2007c).

Standard-sized occluders are available for matching with animal size.

Varying sized occluders can be conveniently obtained commercially (www.doccol.com) with desired tip diameter and silicone rubber coating length. Tip diameter can be selected within a range from 0.17 mm to 0.49 mm and the coating length in a range from 2 mm to 10 mm. This makes it possible to match animal body weight with occluder diameter so as to achieve better results. Although there is not enough available data to make a detailed match chart between occluder size and animal size, a preliminary matching chart is provided by the vendor to guide investigators' selection of occluders, covering animal body weights from 15 to 400 grams.

C. OPTIMIZATION OF SURGICAL PROCEDURES

GENERAL GUIDANCE

The surgical procedure of inducing MCAO models plays an
important role in the stroke outcome variation; and it can be
optimized to achieve better success rates and reduce out-
come variation. Modifications and optimizations have also
been reported concerning the inserted distance of the MCA
occluder, CAA approach versus ECA approach, and sup-
plemental occlusion of proximal arteries.

SPECIFIC GUIDANCE ON KEY SURGICAL STEPS

The inserted distance of the MCA occluder.

**GUIDANCE:** The inserted distance of the occluder is critical to a model's success. For the rat model, the distance from the common carotid artery (CCA) bifurcation is 18-20 mm for a 300 g (Belayev et al. 1996; Lee et al. 2004), and 20-22 mm for a 400 g rat (Lindner et al. 2003). For the mouse model, a distance of 9-11 mm (Dimitrijevic et al.
2007; Yamashita et al. 2006) rostral to the CCA bifurcation needs to be reached.

**SUPPORTING DISCUSSION:** It has been reported that different insertion distances produce significant differences in infarct size (Zarow et al. 1997). Over-insertion may cause a rupture of the anterior cerebral artery (ACA) and subsequent SAH whilst insufficient insertion may not be able to block the back-flow from the anterior communicating artery, leading to incomplete occlusion of the MCA.

*In vivo confirmation of MCA occlusion.*

**GUIDANCE:** A reduction in regional cerebral blood flow (rCBF) of at least 75% from baseline is generally accepted as an indicator of successful MCAO (Schmid-Elsaesser et al. 1998).

**SUPPORTING DISCUSSION:** Because of the anatomic variation of carotid arteries between individuals and between strains (Dittmar et al. 2006; Oliff et al. 1995a; Oliff et al. 1995b), and inaccuracies in measuring the inserted distance, investigators often use LDF to instantly confirm successful occlusion of the MCA. Due to neck movement or artery wall retraction, the occluder may dislodge from its original location if it is not properly affixed against the arterial wall. Occluder dislodgement can result in premature reperfusion and SAH. For reducing occluder dislodgement, investigators have used various techniques. The smooth nylon surface of the occluder at the affixation position can be made serrated to increase traction. A microclip with proper biting force or a tight knot applied onto the artery and the serrated occluder section may help reducing dislodgement.

*CAA approach versus ECA approach.*

**GUIDANCE:** The ECA approach is a better choice for transient MCAO because it maintains the anatomic integrity required for reperfusion. The CCA approach may, on the other hand, be a simpler surgical procedure for permanent MCA occlusion.

**SUPPORTING DISCUSSION:** When inducing MCA occlusion, the occluder may be introduced into the internal carotid artery (ICA) via a cut in the CCA (the CCA approach) or a cut in the external carotid artery (ECA). Most intraluminal models that appear in the literature were induced through the ECA approach. Some stroke investigators introduced the occluder through an arteriotomy of the common carotid artery (Wetzel et al 2008; Xi et al 2004). The CCA approach changes the dynamics of cerebral blood flow when the occluder is withdrawn for reperfusion because blood flow will enter only from the contralateral side through the Circle of Willis. Changes in proximal blood supply may also affect infarct volume and model consistency. Studies have

shown that occluding additional proximal arteries along with MCA can achieve a larger and more consistent infarct (Woitzik et al 2006).

*Supplemental occlusion of proximal arteries*

**GUIDANCE:** Supplemental occlusion of proximal arteries (PTA and/or CCA) decreases infarct volume variation.

**SUPPORTING DISCUSSION:** When the MCA is being occluded, there may be residual blood flow to the MCA territory, which causes insufficient occlusion of the MCA. Residual blood flow could come from the anterior and posterior communicating arteries (AComA and PComA) of the Circle of Willis, the ICA itself, or from leptomeningeal anastomoses on the cortical surface (i.e., collateral supply). Blood flow can also reach the MCA cortex indirectly by external carotid collateral flow through the pterygopalatine artery. Applying a vessel clip on the CCA can increase infarction volume by reducing the residual blood flow through the ICA, especially when smaller filaments are being used (Tsuchiya et al 2003). A more recent study has shown that blocking pterygopalatine artery (PTA) blood flow decreases infarct volume variation (Chen et al 2008). However, from an anatomical perspective, these surgical modifications will not be effective in reducing the residual flow originating from AComA, PComA, or leptomeningeal anastomoses. A more practical option is to use optimized, silicone rubber-coated, standard-sized monofilaments, which match animal body weight, to induce MCA occlusion.

# 6. THE TAMURA MODEL

In 1981, Tamura described a rat model of middle cerebral artery occlusion (Tamura et al 1981a; Tamura et al 1981b) which can induce either permanent or temporary occlusion of the MCA. The former could be achieved by direct electrocoagulation of a section of the MCA whereas the latter by either microclip application or artery ligation/retraction by a nylon suture or a rigid wire. In recent years, infarct variation with the intraluminal models has been noticed (Chen et al 2008; Shimamura et al 2006a), and has become a concern in preclinical neuroprotective trials, especially with suboptimal models (Savitz 2007). Using just one rodent model may not be sufficient for screening neuroprotective candidates in preclinical stroke trials. Therefore, the Tamura model may serve as a supplemental or alternative approach for validating neuroprotective efficacy in rodents.

# A. ABOUT THE MCA OCcludER

**GUIDANCE**
*The MCA occluder and occlusion mechanism play no role in the Tamura model. A careful selection of microclips will be necessary for reducing artery wall mechanical injury.*

## SUPPORTING DISCUSSION

Similar success rates and stroke outcome consistency can be achieved with different occluders and occlusion techniques, such as electrocoagulation, applying a microclip, or suture ligation. Model success rate, mortality rate, and infarct variation will differ in response to changes of occlusion site, occlusion extensiveness, ischemia duration, CCA occlusion, and the animal's blood pressure.

Microclips with a biting force not greater than 15 g are usually used. The size, weight, and biting force of the microclip are important for a successful Tamura model. Not all microclips are suitable for rodent MCA occlusion. Microclips that have been used in this model include Codman Sundt AVM microclip #1-3, Zentype microclip, and Scoville Lewis clip.

# B. SURGICAL OPTIMIZATION

## GUIDANCE

Ensuring a complete occlusion of the MCA is a necessary step for this model. In addition, the occlusion site and extensiveness must remain consistent. In the transient MCAO model, simultaneous occlusion of either common carotid artery increased the model success rate (Coert et al 1999). On the other hand, MCAO combined with bilateral CCAO should be used with caution because of a higher incidence of mortality. Moreover, transient MCAO by the Tamura model is often suboptimal and should be used with caution.

## SUPPORTING DISCUSSION:

Visual inspection under a stereo microscope is commonly used for this purpose. Even with an ensured occlusion through transection of the MCA, infarct consistency still largely depends on the length of coagulated MCA (Bederson *et al* 1986b).

Bederson *et al* first reported (Bederson *et al* 1986b) the correlation of success rate with the anatomic location where the MCA is occluded. In their experiments, a 100% success rate was achieved with 3 or 6 mm occlusion of the MCA beginning proximal to the olfactory tract. 1-2 mm occlusion of the MCA from its origin, at the olfactory tract, or lateral to the inferior cerebral vein, however, only produced infarction in 13%, 67%, and 0% of rats, respectively.

When assessed at 3 days post surgery, one hour MCAO only caused cerebral infarction in 40% of rats; with simultaneous occlusion of ipsilateral or bilateral CCAs, the suc-

cess rate reached 60% and 75%, respectively. A higher success rate was also observed when the duration of MCA occlusion increased. One hundred percent success rate was observed with permanent MCAO plus bilateral CCAO.

Chen *et al* reported (Chen *et al* 1986) that the mortality rate could reach 60% when the CCAs were permanently ligated bilaterally; the high mortality could be reduced to 7% if the contralateral CCA was released after 60-min of occlusion.

Although transient direct MCA occlusion with subsequent reperfusion is possible by applying a microclip on, or suture tying, the MCA, the implementation of these techniques demands extremely delicate surgical skills, especially in mice. Consequently, this model has gradually become less popular since the emergence of the intraluminal model of transient MCAO (Koizumi *et al* 1986), which is relatively easier to perform and does not require a craniotomy.

# C. PURE CORTICAL INFARCTION VS. SUS COMBINED INFARCTION

## GUIDANCE

Pure cortical infarcts in this model produce very mild and inconsistent functional deficits and are therefore not suitable for functional recovery evaluation. Permanent occlusion at a site proximal to the lenticulostriate branches (pMCAO) produces a larger infarct with more persistent functional deficits (Roof *et al* 2001), and may be preferable for assessing a robust protective effect of therapeutic agents for stroke.

## SUPPORTING DISCUSSION

The site of MCA occlusion has also been shown to influence the severity and consistency of histologically-revealed damage as well as functional deficits. Occlusion of the MCA lateral to the olfactory tract produces a pure cortex infarction because the basal ganglia blood supply from the lenticulostriate branches is spared; occlusion of the MCA at its origin produces a combined infarction including both cortex and basal ganglia. Microclips and retraction/release methods are used for transient MCAO/reperfusion and produce a pure cortical infarct due to operational limitation. Electrocoagulation necessarily produces permanent occlusion; it can be applied at various sites along the MCA to produce a variety of lesions from a pure cortical infarct to combined infarcts of both basal ganglia and cortex.

# D. INFARCT VOLUME VARIATION AND OPTIMAL ISCHEMIA DURATION
## GUIDANCE

Transient MCA occlusion of 30-min by the Tamura model is too mild to produce a brain infarct, but selective neuronal damage in the striatum and subcortex areas in the ipsilateral side could be observed (Yang et al 2001). A 3-h occlusion time is preferable for transient cortical ischemia because of increased consistency. Infarct volume consistency could be improved with supplemental CCA occlusion. Similar results have been reported with different occlusion methods.

## SUPPORTING DISCUSSION

Improved infarct consistency has been reported with simultaneous occlusion of the common carotid arteries (Coert et al 1999). For example, the pSDM for 1-h MCAO was 200%, 1-h MCAO plus ipsilateral CCAO was 133.5%, and 1-h MCAO plus bilateral CCAO 100.9%. The pSDM for 3-h MCAO plus bilateral CCAO was 59%. The best consistency was observed with permanent MCAO plus bilateral CCAO with the pSDM being 43.75%.

A pSDM range of 10% to 160% has been reported when using a Zen-type microclip for direct MCA occlusion in rats. Margaill et al achieved excellent consistency in rats, with a pSDM of 10% for striatum infarcts and 15-16% for cortex infarcts in transient MCAO of 60-90 min (Margaill et al 1996). However, in a 1-h MCAO model, David et al reported significant variation with the pSDM being 68% (David et al 1996). Morikawa et al reported an even higher pSDM of 160% for cortex infarcts and 83% for striatum infarcts with a 2-h transient MCAO (Morikawa et al 1992). Using a Zen-type microclip for MCA occlusion in mice seems to result in less variation than in rat models. Kitagawa et al achieved a pSDM of 10% for permanent MCAO, and of 45.65% for 60-min transient MCAO (Kitagawa et al 2004).

When proximal arteries were occluded in addition to microclip occlusion of the MCA, a pSDM range of 16% to 119% could be achieved. Using a Sundt microclip for direct MCAO plus bilateral CCAO, Buchan et al (Buchan et al 1992) achieved pSDMs of 119%, 86% and 16% for 1-h, 2-h, and 3-h transient MCAO respectively, when combined with the same duration of contralateral CCAO and permanent ipsilateral CCAO, in normotensive rats. A pSDM of 32% could be reached for 24-h permanent MCAO combined with permanent bilateral CCAO. In a 3-vo transient MCAO model with all three vessels released at the same time after a period of occlusion, the pSDM was 38% (Schielke et al 1999) for 3-h transient MCAO, and 32% (Coert et al 2003) for 2-h transient MCAO. In hypertensive rats, a 90-min transient MCAO combined with permanent occlusion of the ipsilateral CCA can produce a consistent infarct volume with a pSDM of 25% (Colbourne et al 2000).

Using suture tying methods in rats, the pSDM range has been reported as being from 13% to 99%. Selman et al reported the pSDM in a 1-h transient MCAO model to be 99% (Selman et al 1994). Better consistency could be achieved by increasing the duration of MCA occlusion. Permanent MCA ligation combined with permanent ipsilateral CCA ligation and transient 60-min occlusion of the contralateral CCA (Chen et al 1986) produced a pSDM of 19%. Infarct variation coefficients of 13% have been reported (Drummond et al 1995) with 3-h transient MCAO in hypertensive rats.

For MCA cauterization and permanent cut methods, a pSDM range of 6.6% to 149% has been reported. Morikawa reported a pSDM of 53% for cortex infarcts and 34% for striatum infarcts (Morikawa et al 1992). In a permanent distal MCAO model with ipsilateral CCA occlusion, Brint et al also reported (Brint et al 1988) a pSDM of 16-149% in Wistar rats and 6.6-35% in spontaneously hypertensive rats, which was associated with a more severe infarct in Wistar rats.

A pSDM range of 12.5% to 50% could be reached in a 3-vo Tamura model. Yanamoto et al evaluated a 3-vo model both in normotensive rats and mice, in which the MCA was cauterized and cut permanently, along with temporary bilateral CCAO (Yanamoto et al 2003). When the CCAs were released after 60-min, the pSDM reached 50% in rats and 24% in mice. When the CCAs were released at 2-h post occlusion, the pSDM in rats was reduced to 12%; this is an improvement over their earlier work which described (Yanamoto et al 1998) a pSDM of 22% in a 3-vo transient MCAO of 2-h in normotensive rats (all three vessels were released after 2-h occlusion).

# 7. INFARCTION VOLUME MEASUREMENT

## A. METHODS FOR INFARCTION VISUALIZATION

Measuring infarct volume evolution is time sensitive and methodology dependent. Tissue processing for histopathological staining may produce significant volume variation. Definitive determination of cerebral infarct is made by microscopic examination of hematoxylin and eosin (H&E) stained brain sections. Infarcted brain tissue appears as a sharply delineated pan-necrotic area on H&E stained brain sections (Garcia et al 1993). On H&E stained brain sections ischemia-induced neuronal morphological changes can be detected within a few hours after MCA occlusion while it usually needs 24-h for these ischemic changes to mature into a well-developed infarct. There are other more sensitive staining methods that can detect ischemic injury
as early as 15-min post MCA occlusion. These staining methods include the arginophilic III staining (Czurko and Nishino 1993; Liu and Guo 2000a) and the immunohistochemical staining of microtubule-associated protein 2 (MAP2) (Pettigrew et al 1996). The early infarct area revealed using the above-mentioned pathological methods does not usually have enough contrast when compared with adjacent non-ischemic tissue. This makes it difficult for direct macrometric measurement of infarct volume. Alternatively, the macrometric measurement of infarct volume can still be achieved after microscopic delineation of the infarct area (Liu and Guo 2000b). The above mentioned methods also require tissue fixation followed by a complex staining process, which may produce 7-12% variation of hemisphere volume (Overgaard and Meden 2000). Therefore, a standard tissue processing protocol for these methods is needed for reducing variation. Currently, direct macrometric measurement of brain infarction is most often conducted by using 2,3,5-triphenyltetrazolium chloride (TTC) to stain fresh brain sections. The TTC staining method is able to offer a reasonably sharp contrast between infarcted and normal areas as early as 3-h in rats, and 12-h in mice. It is relatively simple to conduct and is widely accepted by most stroke investigators.

## B. DIRECT VISUALIZATION OF INFARCT ON TTC STAINED FRESH BRAIN SECTIONS

### GUIDANCE

TTC staining is the most widely used technique to identify infarcted versus viable tissue. It is not selective for brain tissue or cell types. A brain matrix or vibratome is necessary for providing clean cut sections. The extent of brain infarction is optimally seen between 24-36 h post ischemia by the staining of fresh brain sections. Species differences in mitochondrial dehydrogenases may account for differences in the times at which infarction can become apparent (see below). In vivo TTC staining should be used only for transient ischemic models in its reperfusion stage after excellent reperfusion has been ensured. Better contrast and infarct boundary delineation may be obtained with use of lower TTC concentration.

### SUPPORTING DISCUSSION

TTC serves as a proton acceptor for many pyridine nucleotide-linked dehydrogenases (such as succinate dehydrogenase); it is reduced by these enzymes in viable brain tissue into a red, lipid-soluble formazan, while infarcted or non viable tissue remains unstained (Bederson et al 1986a; Liszczak et al 1984). The TTC method requires the brain to be sectioned into several thin parallel sections with even

surfaces for infarct volume calculation. It has been reported that there is good correlation between TTC, H&E (Bederson et al 1986a; Lundy et al 1986), and cresyl violet staining (Tureyen et al 2004). Although TTC staining is widely used for infarct volume measurement, there are some issues that need to be considered regarding its use.

Macrophage/glia infiltration may confound the staining results after 36-h post-ischemia. Infiltrating cells may cause staining in infarcted tissue. For example, 36-h after stroke, macrophages and glial cells infiltrate infarcted areas, and result in tissue TTC staining, which would not have been evident at an earlier time point (Liszczak et al 1984). Another issue that needs to be considered is the species difference of mitochondrial enzymes (Stewart et al 1998). For example, ischemic injury can be visualized as early as 3 hours after stroke in rats (Bederson et al 1986a; Liu et al 2004), but may require at least 12 hours in mice.

TTC is able to pass the blood-brain barrier, which allows *in vivo* staining (Isayama et al 1991). However, such *in vivo* staining relies on the regional cerebral blood flow (Dettmers et al 1994), and may only be suitable for transient cerebral ischemia at the reperfusion stage; it cannot be used in permanent cerebral ischemia (Benedek et al 2006).

The TTC staining process is affected by several factors, such as TTC concentration, staining duration, and incubation temperature. A methodology paper (Joshi et al 2004) demonstrated that staining with lower TTC concentration (0.05-0.1% versus 0.6%) at 37°C for 30-min could reduce non-specific staining and improve contrast between infarcted and normal tissue, and hence provide better delineation of infarct boundaries.

## C. DIGITAL METHODS FOR DEFINING THE INFARCTED AREA

### GUIDANCE WITH SUPPORTING DISCUSSION

The traditional way of acquiring a digital image of brain infarct is to digitalize the brain section through a stereoscope equipped with a macro lens. TTC-stained brain sections can also be scanned into digital files for automated infarct recognition (Goldlust et al 1996). Manual delineation of the infarct area may be needed if the contrast is insufficient for an automatic infarct selection. Due to field limitation of the regular objective lens, additional optical modification may be required in order to be able to view the entire brain section with a regular microscope. For volume calculation, the infarct area must have enough contrast against the non-infarcted area that it can be distinguished from its surrounding areas. Infarct area can be measured using imaging analyzing software such as Image Pro Plus(Liu et al 2006), Adobe Photoshop (Horita et al 2006), NIH image
J (Tureyen et al 2004), or other appropriate image processing programs. If the contrast is excellent, as it usually appears on TTC stained sections, the infarct area can be automatically selected and calculated based on color differentiation. With spatial calibration the infarct area can be expressed in real measurement units (e.g., mm³).

# D. CALCULATION OF INFARCT VOLUME

## GUIDANCE

When comparing infarction volumes at different time points, cerebral edema and infarct shrinkage should be corrected for.

## SUPPORTING DISCUSSION

Ischemic infarction evolution involves different temporal-spatial pathological processes that may influence infarction volume measurement. Studies on the natural progress of infarct evolution show significant differences in infarction volume between early and late time points (Gaudinski et al 2008; van der Worp et al 2005). Cerebral edema is more severe 2-3 days after acute stroke. Edema may significantly increase the brain tissue volume as well as the directly measured infarct volume. On the other hand, when an infarct has been evolving for one week, it will begin to shrink because of attenuated edema, tissue loss, and scar contraction. When comparing infarction volumes at different time points, cerebral edema and infarct shrinkage should be adjusted. In this situation, a corrected infarction volume against edema or shrinkage (Leach et al 1993; Lin et al 1993; Swanson et al 1990) will be more suitable.

# E. CALCULATION FORMULAE FOR INFARCTION VOLUME

The following formulae can be used for calculating the corrected infarction volume for both edema and shrinkage.

$$ \text{Corrected infarct area} = \text{measured infarct area} + \text{area of contralateral corresponding structure} - \text{area of ipsilateral corresponding structure} \text{ (Swanson et al 1990)} $$

$$ \text{Corrected infarct area} = \text{measured infarct area} \times \text{area of contralateral corresponding structure} / \text{area of ipsilateral corresponding structure} \text{ (Leach et al 1993)} $$

Infarction volume for continuous macrosections can be calculated as $\Sigma(thickness \times 1/2)$ (corrected infarct area of a section's rostral surface + corrected infarct area of a section's caudal surface)

If the infarct volume calculation is based on thin microsections (5-10 µm thickness) at fixed interval, the formula will look like this:

$$ \text{Infarct volume} = (\text{interval} + \text{slide thickness}) \times (\Sigma \text{ (corrected infarct area)} - 1/2 \text{ corrected infarct area of the first slide} - 1/2 \text{ corrected infarct area of the last slide}) $$

# F. MEASUREMENT OF INFARCTION VOLUME VARIABILITY

## GUIDANCE

Use relative volume for comparison between studies and indicate infarct volume variation.

## SUPPORTING DISCUSSION

Experimental stroke models are performed with one or more of a variety of possible subject animals according to the needs of the study design. In addition, researchers measure infarct volume using different units and at various time points post ischemia. This situation generally precludes direct comparison of infarction volumes between studies. Standard deviation (SD) reflects the variability in a set of data while standard error of mean (SEM) reflects the accuracy of a mean value. Therefore the SD of infarct volume reflects the stroke model outcome variation. SD can be normalized to its corresponding mean as a Percentage of SD to Mean (pSDM). Some authors in the cited papers expressed data as mean ± SEM; in these cases we have converted SEM to SD according to the formula $SD = SEM \times \text{SQRT N}$.

# 8. FUNCTIONAL EVALUATION

## A. THE TIMING OF FUNCTIONAL EVALUATION

### GUIDANCE

The natural process of functional recovery should be considered in functional evaluation after experimental focal stroke. Evaluation should be conducted at the same post-occlusion time point in all groups but not during the 6-12 hours post-occlusion because at this time accelerated functional recovery occurs. Maximal sensitivity for functional evaluation can be achieved between 2-6 hours post-occlusion.

### SUPPORTING DISCUSSION

The extent of functional recovery after stroke is dependent on time, age and environmental factors (Buchhold et al 2007). Some functions recover faster and better than others. The most severe sensorimotor deficits can be observed at 2-6 hours post stroke with a fast recovering
speed being observed between 6-12 h post-MCAO (Reglo-
di et al 2003). For validating neuroprotective efficacy, func-
tional tests with a slow or absent natural recovery process
may be most appropriate, such as forelimb flexion, gait
disturbance, and lateral resistance (Reglodi et al 2003).
The well-known "circling phenomenon" can be observed as
soon as the animal is fully recovered from anesthesia, but
may not be apparent when evaluated at 24 hours in some
stroke models (Erdo et al 2006) despite significant infarct
volume maturation at this time. In a permanent MCAO
model resulting in cortical infarction, it has been reported
that most young rats (3-4 mo) do not show "circling" when
evaluated on day 2 post ischemia (Buchhold et al 2007).
Hence, such a highly time-dependent motor deficit may not
be suitable for preclinical neuroprotection stroke studies. In
order to achieve a better sensitivity in detecting neuropro-
tective efficacy, MCAO models of moderate severity should
be used and appropriate functional tests should be con-
ducted between 2-6 hours post ischemia because function-
al deficits usually reach maximum severity at this time. For
confirming robust neuroprotection, functional tests that
have a slow recovery pattern may be more appropriate.

B. EVALUATION SYSTEMS FOR NEU-
ROLOGICAL FUNCTIONAL DEFICITS

GUIDANCE

Select an appropriate evaluation system to cover important
functional deficits. It is reasonable to include more tests to
cover different functional deficits and subsequently analyze
both the total score and individual scores because each
function has a different recovery pattern. Functional tests
that have a slow recovery pattern may be most suitable to
confirm a neuroprotective effect.

SUPPORTING DISCUSSION

Behavioral changes after ischemic stroke can be evaluated
using specially designed scales. Many scales are available
for the detection of ischemic injury, but not all scales can
be used for the validation of an intervention's neuroprotec-
tive capability. To qualify for neuroprotection studies, the
neurological scale must be able to detect the major ische-
mia-induced behavioral changes, including motor, sensory,
motion coordination, spontaneous activity, reflexes, con-
sciousness, and alertness changes. Bederson' 4-point
scale (Bederson et al 1986b), modified Bederson's scale
(Becker et al 2001; Zausinger et al 2000), and Rogers' 8-
point scale (Rogers et al 1997), although frequently used,
are primitive measurements of motor deficits. It may be
more appropriate if these scales are merely used for con-
firming a successful occlusion of middle cerebral artery
after completion of surgery. For a more informative func-

tional assessment, more complex evaluation systems like
the 18 and 42 point scales should be considered (Chen et
al 2001; Reglodi et al 2003). Although several functional
tests have been developed to provide an effective neuro-
logical evaluation scale for preclinical neuroprotection stu-
dies (Buchhold et al 2007; Reglodi et al 2003; Schallert
2006), there are no guidelines regarding their use.

C. ANALYSES FOR NEUROLOGICAL FUNCTIONAL DEFICITS

GUIDANCE

Ensure a blind method is adhered to for conducting the
evaluation process. Analyze both the total score and indi-
vidual scores. When a complex battery of tests is being
used, stratified analysis of functional deficits will be prefer-
able because the changing pattern will be different in func-
tional deficits. When indicated, use non-parametric statis-
tical methods for data analysis.

SUPPORTING DISCUSSION

During the evaluation process, behavioral scores are given
by the examiner based on the examiner's observation and
understanding of the tests; therefore, these scores are sub-
ject to the examiner's bias. Adapting a blind method for
functional evaluation will be necessary for reducing bias in
preclinical neuroprotective trials. Moreover, the data set
obtained from scaled neurological evaluation may not al-
ways conform to a normal distribution, especially when the
sample size is small. Non-parametric statistical analyses
should be used if the data can't pass a normality test. For
example, one should use a Mann-Whitney U test for two
group comparison and a Kruskal-Wallis analysis of ranks
for multiple group comparison.

9. ANESTHETICS

GUIDANCE

When designing a preclinical study for neuroprotection, the
protection provided by anesthetics should be taken into
account. When neurotransmitters or neuroplasticity are the
main foci of a study, anesthetics such as urethane, which
do not disturb the action of neurotransmitters should be
used. Fasting animals should be utilized in the experimen-
tal design of neuroprotection studies though caution should
be used to reduce hypoglycemia-related mortality when
fasting small rodents (mice, gerbils).

SUPPORTING DISCUSSION
Many commonly used anesthetics have neuroprotective effects against cerebral ischemic injury. These anesthetics include isoflurane (Kawaguchi et al 2004; Xiong et al 2003), sevoflurane (Nakajima et al 1997; Payne et al 2005), desflurane (Erdem et al 2005; Tsai et al 2004), halothane (Haelewyn et al 2003), xenon (David et al 2008), nitrous oxide (Abraini et al 2004; Haelewyn et al 2008), barbiturates (Warner et al 1996), propofol (Bayona et al 2004), ketamine (Proescholdt et al 2001), and the local anesthetic lidocaine (Siniscalchi et al 1998; Weber and Taylor 1994)..

Most anesthetics have been found to potentiate the inhibi-
tory activity of the g-aminobutyric-acid (GABA)-A receptor.
Volatile anesthetics are also antagonists of N-methyl-D-
aspartate (NMDA) and amino-3-hydroxy-5-m ethyl-4-
isoxazol-propionic acid (AMPA) receptors, and openers of
K+ channels (Grasshoff et al 2005). Therefore, the working
mechanism of anesthetics needs to be considered during
the experimental design of neuroprotection studies (Maggi
and Meli 1986; Sceniak and Maciver 2006). When an ex-
pected neuroprotection is likely via the opening of K+
channels, volatile anesthetics may have compounding ef-
fect and should be avoid when possible.

Hyperglycemic effects only occur in fed animals, and thus
can be eliminated by fasting animals 18-24h before expe-
imentation. Xylazine is an α2-adrenergic agonist, which
decreases plasma insulin level and induces hyperglacemia
(Greene et al 1987; Thurmon et al 1984). Hence, xylazine
should be avoided when blood glucose level becomes a
concern in experimental design. In addition to xylazine's
hyperglycemic effects, some commonly used volatile anes-
thetics, such as isoflurane and halothane, also may cause
a rapid increase in blood glucose levels (up to 230 mg/dl or
12.6 mmol/L) within 20min of induction. Ketamine/xylazine
can result in hyperglycemia reaching 290 mg/dl (15.9
mmol/L) (Saha et al 2005)..

10. TEMPERATURE

A. THE NECESSITY OF MONITORING TEMPERATURE

GUIDANCE

Controlling animal body temperature in a normal range is necessary for eliminating the protective effect of hypothermia and potential harmful effect of hyperthermia (Zaremba 2004).

SUPPORTING DISCUSSION

Brain temperature during hypoxia affects brain metabolism
significantly (Winn et al 1981). Hypothermia reduces (Flo-
rian et al 2008; Miyazawa et al 2003; Ohta et al 2007) and

hyperthermia exacerbates (Kim et al 1996; Noor et al 2003; Noor et al 2005) ischemic brain injury, hence, fluctuation in animal body temperature will increase the variability of stroke outcome. Ischemia itself also affects post-ischemic temperature regulation, which in turn influences the extent and severity of brain injury and functional deficits (Colbourne et al 2000)..

B. MONITORING BRAIN/CORE TEMPERATURE

GUIDANCE

Various methods may be used for monitoring body temper-
ature in stroke animal models. The simplest way of monitor-
ing temperature is by placing a temperature probe in the
rectum of the anesthetized animal. Monitoring brain or peri-
cranial temperature may be performed with caution in some
experiments when a difference between brain and rectal
temperatures is predicted. Temperature monitoring should
commence before inducing anesthesia and there is also a
need to monitor temperature after surgery.

SUPPORTING DISCUSSION

Monitoring rectal temperature assumes that there are no,
or at least there are predictable, differences between brain
and rectal temperatures. However, brain temperature may
actually be considerably different from rectal temperature
during the ischemic period (DeBow and Colbourne 2003;
Marion 2004; McIlvoy 2004; Nussmeier 2005). Pericranial
temperature can be monitored by placing a subcutaneous
needle thermistor adjacent to the skull in the temporal
muscle, (Xiong et al 2003; Yonekura et al 2004) although it
should be noted that this measurement is very sensitive to
small changes in needle position. Alternatively, for experi-
ments studying the effects of temperature changes during
cerebral ischemia, a thermistor can be inserted directly into
the brain (DeBow and Colbourne 2003; Menzel et al 1998).
However, the latter method involves a relatively complex
and invasive surgery to insert the brain probe, and runs the
risk of causing brain injury and infection.

Hyperthermia or hypothermia can occur because of the
surgery, anesthetic, ischemia, and/or unexpected infection.
The use of telemetric temperature probes for monitoring
post-ischemic body temperature has a significant advan-
tage in its capacity to measure temperature continuously in
conscious animals. In addition, telemetry can be used with
an automated feedback system for temperature control.
Disadvantages of this method are the high cost of teleme-
tric systems and the need for additional surgery to implant
probes.
## C. MAINTAINING BRAIN/CORE TEMPERATURE

### GUIDANCE

Maintaining core temperature within an appropriate range during ischemia can be achieved by using a water heating pad, electric heating blanket, heat lamp and/or heating fan. PID temperature controller equipped heating devices provide fast response and precise temperature control. Electric blankets are not recommended if a telemetric system is being used as they may interfere with the probe signal. Maintaining body temperature after surgery is necessary. This may be done by placing animals in a humidified warm chamber for a few hours.

### SUPPORTING DISCUSSION

Since the thermal conduction of a water heating pad may not be rapid, an overhead incandescent lamp or heating fan may serve as a complementary heating source to further control temperature. It must be taken into consideration that the overhead heating source may interfere with the operation by heating the surgical tools and the operator's hands. Temperature control when using electric heating pads and blankets can be improved by using a proportional integral derivative (PID) temperature controller to allow ex vivo or in vivo feedback from the sensor that monitors heating pad or animal body temperature. The electric current to the heating pad can be either direct current (DC) or alternating current (AC), depending on the experimental design and budget. DC powered heating devices have less electric noise and are well suited for electrophysiological studies. AC powered heating devices may be used in most preclinical stroke trials that do not need electrophysiological monitoring.

Maintaining body temperature after surgery is just as important as during the ischemia period because the full recovery of animal body temperature regulation needs time (Chang et al 2008; Jia et al 2006). The most popular method of maintaining body temperature is by using a warm chamber that keeps the environmental temperature at 28-32°C. The considerable advantage of the thermo-controlled temperature regulation system with an in vivo feedback system, as described by Colbourne et al (Colbourne et al 2000), is that the investigator can regulate the temperature of conscious animals with precision during the post-ischemia period. Precise temperature regulation can be achieved with the automated telemetry system, which uses fine water mist with overhead fans for cooling and infrared lamps for heating.

## 11. MECHANICAL VENTILATION AND BLOOD GAS/GLUCOSE MONITORING

### A. PROPER USE OF MECHANICAL VENTILATION

#### GUIDANCE

The importance of using mechanical ventilation should be determined by the anticipated impact of the surgical/anaesthetic procedure on respiratory function. The potential confounding effects from respiratory functional deficits can be minimized by the use of mechanical ventilation. Unnecessary use of mechanical ventilation should be avoided when a particular MCAO model is not likely to cause respiratory problems.

Ventilation may be needed when the operation lasts long (>1 hour) and when the ischemia affects brain stem function. A mixture of 30%:70% (O₂:N₂ or N₂O) may be used for preclinical stroke trials combined with individualised adjustment of ventilator parameters. The concentration of inspired oxygen and ventilator parameters (tidal volume, airway pressure, respiratory rate, inspiratory/expiratory duration) can be roughly determined by a pilot experiment with periodic measurements of arterial blood gases. The respiratory rate and stroke volume can be set differently in accordance with the different "dead space" of each ventilator and anesthetic circuit.

### SUPPORTING DISCUSSION

Hypoxia is injurious to the CNS, especially the adult brain. Normobaric hyperoxia (Singhal et al 2002) and hyperbaric oxygen treatment (Iwatsuki et al 1994; Takahashi et al 1992; Wallsh et al 1986) have been demonstrated to be neuroprotective during ischemia and reperfusion, but also can have deleterious effects on the normal and injured central nervous system (CNS) (Bostek 1989; Bulte et al 2007; Diringer 2008). Similarly, as carbon dioxide is a potent cerebral vasodilator and causes increased cerebral blood flow (CBF) (Kontos et al 1977a; Kontos et al 1977b; Rusyniak et al 2003), hypercarbia may have a protective effect during ischemia and reperfusion (Vornov et al 1996). On the other hand, extracellular acidosis caused by hypercapnia may inhibit neuronal functions (Velisek 1998) and cause adenosine triphosphate (ATP) depletion (Yamamoto et al 1997). In addition to the above, intubation and mechanical ventilation are commonly used to improve control of blood oxygen and carbon dioxide levels. However, the intubation procedure itself and control of the mechanical ventilation process are technically demanding and may cause tissue damage even in experienced hands. The use of mechanical ventilation
tion will mostly depend on the nature of the experiments. If the experiment is not likely to cause respiratory failure, intubation and mechanical ventilation may not be necessary.

Since respiratory function might be suppressed by anesthesia, endotracheal intubation and mechanical ventilation may be necessary in order to maintain blood gases within the normal range. This is especially relevant during long operations (>1 hour) and when the ischemia affects brain stem function. The components and concentrations of the inspired gas are essential for maintaining arterial blood gases within a normal range when the airway is secured. Oxygen and nitrous oxide are traditionally used in a mixture of 30%:70% (O₂:N₂O) in rodent stroke models. Because nitrous oxide has been shown to be neuroprotective in ischemia-induced brain injury (Abraini et al 2004; Haelewyn et al 2008), it may be preferable to use nitrogen mixed with oxygen. Since hyperoxia has been shown to have a neuroprotective effect in brain ischemia (Liu et al 2006; Singhal et al 2005; Singhal 2007), oxygen concentration and pressure in the inspired air should be controlled at a stable level so as to avoid hypoxia and hyperoxia. Therefore, a mixture of 30%:70% (O₂:N₂) may be used for preclinical stroke trials combined with individual adjustment of ventilator parameters. Note, however, that substituting N₂ for N₂O may slow induction and recovery times, and will generally require that the concentration of volatile anesthetics be adjusted upwards.

Several studies (Bottiger et al 1999; Olsson et al 2003; Yang et al 1997; Yonekura et al 2004) have shown that with an inspired gas mixture of 30% O₂ and 70% N₂O, the pre-ischemia levels of partial oxygen pressure (PaO₂), partial carbon dioxide pressure (PaCO₂), and pH varied from 93.4 ± 21.1 to 208 ± 45 mmHg, from 28.1 ± 4.6 to 40 ± 5 mmHg, and from 7.12 ± 0.04 to 7.39 ± 0.08, respectively. One likely reason for these large variations may be that different tidal volume and respiratory rates were used throughout these studies. For example, the respiratory rate and the stroke volume were set at 120 breaths/min and 0.25 ml in the studies of Olsson et al (Olsson et al 2003) whilst they were set as 130 breaths/min 0.7 ml in those by Sheng et al (Sheng et al 1999). In addition, the normal resting tidal volume of animals ordinarily will increase, and the respiratory rate will decrease, in proportion to increases in body weight.

## B. MONITORING GLUCOSE

### GUIDANCE

It is necessary to monitor blood glucose levels before, during and after ischemia, especially in models causing severe brain damage, or in certain newly acquired genetically modified strains. A glucose meter may be preferable to the

integrated glucose measurement function of a standard blood gas analyzer. During the post-surgery stage, hypoglycemia can be prevented by proper care. An animal's appetite, food consumption, and body weight should be monitored, and supplemental administration of glucose by gavage or intraperitoneal injection may be needed. Hyperglycemia can be prevented by fasting the animal overnight before surgery. Any observed hyperglycemia is usually not treated, but it may be used as a criterion for subgrouping or excluding animals in data analyses.

## SUPPORTING DISCUSSION

Since hyperglycemia can cause exacerbation of ischemic damage, glucose should be routinely measured during experimental stroke (Lovblad et al 2003; Parsons et al 2002). Many commonly used volatile anesthetics such as isoflurane and halothane cause a rapid increase in blood glucose (Saha et al 2005). Some transgenic animals (Rajkumar et al 1995; Rajkumar et al 1996) might have congenital diabetes or have a tendency to suffer hyperglycemia after an ischemic insult. In contrast, the loss of appetite or inability to access food may also cause hypoglycemia in animals and may potentially affect survival rates and outcomes, especially in small rodents (mice and gerbils).

For blood glucose assay, a glucose meter may give more precise readings than the integrated glucose measurement function incorporated into a blood gas analyser. In addition, a glucose meter uses much less blood than a blood gas analyser and is usually quicker.

## C. BLOOD SAMPLING FOR BLOOD GAS ANALYSES

### GUIDANCE

Blood sampling is necessary for periodic measurement of arterial blood gas and frequency of measurement should be selected with reference to animal size. Although pulse oximetry for measuring oxygen saturation has been widely used in clinics, its value in middle cerebral artery occlusion (MCAO) models is not clear. It may be considered as an alternative option when blood sampling from mice/gerbils is not possible.

### SUPPORTING DISCUSSION

In larger animals, it is feasible to adjust the stroke volume and the respiratory rate of the ventilator based on the periodic measurement of arterial blood gas. However, frequent blood sampling is not possible in small animals like gerbils and mice, due to their limited blood volume. In our experience, two blood samples (0.08ml per time) can be taken in mice using a capillary tube without affecting the survival
rate and ischemic outcome. The first sample could be tak-
en 10 minutes after ventilation and before ischemia; the
second sample can usually be taken right after ischemia
has ended. The first sample is preferably used to determine
whether the ventilator is set properly because physiological
parameters may change significantly in the post ischemia
period (Bottiger et al 1999).

12. BLOOD PRESSURE

GUIDANCE

Monitoring blood pressure during experiments is needed because blood pressure fluctuation affects stroke outcomes. Blood pressure can be monitored by non-invasive and invasive methods. Use non-invasive methods for experiments that cause minimal blood pressure fluctuation and require a neurological evaluation. Use invasive methods for experiments that require constant blood pressure monitoring. Blood pressure fluctuation due to cerebral ischemia is usually not corrected during the experiment, although it can be used as a guide to anesthetic depth and the concentration of inspired anesthetic gas can be adjusted if appropriate.

SUPPORTING DISCUSSION

In stroke models blood pressure can fluctuate due to anes-
thetic depth and changes in animal body temperature. In
addition, during the ischemic period, blood pressure may
be elevated due to the systemic response in attempting to
maintain a normal brain perfusion pressure. The change of
blood pressure affects regional cerebral blood flow and
hence stroke outcome (Drummond et al 2000; Kawaguchi
et al 2004) and clinical trials (Cole et al 1990; Rordorf et al
1997; Wise 1970; Zhu and Auer 1995). Therefore, blood
pressure should be monitored during experiments, espe-
cially when a significant fluctuation of blood pressure is
expected.

Non-invasive blood pressure monitors are equipped with
tail-cuff devices for artery occlusion and oscillometric pulse
detectors for readings. Because of the limited accuracy and
the relatively long interval (in minutes) between two se-
quential measurements, this method is not suitable for ex-
periments that need constant blood pressure monitoring.

Invasive blood pressure monitoring provides constant read-
ings throughout the experiment. A disadvantage of invasive
blood pressure monitoring, however, is the need to estab-
lish an arterial line to connect to a pressure transducer.
Additionally, when cannulation of the femoral artery is uti-
lized, the cannulating process and the wound associated
with the arterial line may interfere with subsequent neuro-
logical function evaluation because of pain, impaired blood

flow and potential nerve injury in the affected limb (Zlotnik
et al 2008). However, if the arterial line used for blood
sampling is also used for blood pressure monitoring the
problems are minimized.

Blood pressure is a sensitive indicator for assessing anes-
thetic depth, and is also an indicator for ventilation efficien-
cy. Anesthetic dose and ventilation can be modified accor-
dingly before the systemic blood gas changes occur. With
the exception of experiments specifically designed for stud-
ying the effects of blood pressure on brain injury, blood
pressure manipulation is usually not suggested when the
blood pressure fluctuation is a result of an ischemic insult.
In addition, the blood pressure information obtained during
experiments may serve as evidence for exclusion or inclu-
sion of animals for the final analyses.

13. THE IMPORTANCE OF A PILOT STUDY

GUIDANCE

A pilot study should be performed before the implementa-
tion of a preclinical stroke trial. Stroke model success rate,
mortality rate, outcome variation, and sample size should
be determined through the pilot study.

SUPPORTING DISCUSSION

Although information regarding the suture size, coating
length, insertion length, and related surgical procedures is
available in the literature, these parameters may not be
optimal for your own experiment. In addition, the intralum-
minal stroke model demands delicate surgical skill; inexpe-
rienced surgeons need a period of time to command the
necessary skills for producing acceptably consistent re-
sults, which is referred to as the “surgeon’s learning curve”
(Renzulli and Laffer 2005). For these modeling reasons, a
pilot study is needed to find out the optimal parameters for
the MCAO suture and the lab settings for any new study.
The pilot study will also be helpful for study design because
it may provide the closest information for the expected in-
fact variation, success rate, and mortality rate.

14. IMPLEMENTATION OF THE PRECLINICAL STROKE TRIAL

A. ADHERE TO “GOOD LABORATORY PRACTICE”

The implementation of a preclinical stroke trial should be
conducted with high standards so that experimental bias
can be minimized. Some journals have set "Good Labora-
tory Practice" standards for publishing preclinical trials, and
only studies fulfilling these standards will be accepted for publication in these journals. As stated in "Good Laboratory Practice" (Macleod et al 2009a; Macleod et al 2009b; Macleod et al 2009c), a preclinical stroke trial should be conducted with a clear methodology which includes at least the following items,:

* Detailed information on animals used;

* Sample Size Calculation;

* Inclusion and Exclusion Criteria;

* Randomization;

* Allocation Concealment;

* Reporting of Animals Excluded From Analysis;

* Blinded Assessment of Outcome;

* Reporting Potential Conflicts of Interest and Study Funding.

## B. USE APPROPRIATE STATISTICAL METHODS FOR DATA ANALYSES

It is very important to use the correct statistical method for data analyses. Scaled data (such as neurological evaluation and semi-quantitative data) and categorical data (such as mortality rates) should be treated with caution because incorrect statistical methods may lead to invalid conclusions. As discussed in the *Functional Evaluation* section, scaled neurological scores may not always conform to a normal distribution and non-parametric statistical analyses should be used if the data can't pass a normality test. For example, one should use a *Mann-Whitney U*-test (Estevez and Phillis 1997) for two group comparison and a *Kruskal-Wallis* analysis of ranks (Meden et al 2002; Onal et al 1997) for multiple group comparison. The mortality rate is a type of categorical data; therefore, its analysis should use the *Chi-Square* test (Lu et al 2009), not *Student's t*-test (Tang et al 2005).

## 15. FACTORS BEYOND SCIENCE

### A. THE NEED FOR SOPS FOR STROKE MODELS

Stroke model procedures, especially those steps that influence model quality, have not yet been standardized. Investigators in each laboratory implement the stroke model with their own lab-settings. In addition, many models have been conducted using suboptimal procedures. This situation makes the results less comparable between laboratories. Standard operational procedures (SOP) for stroke models, if available, may help to improve inter-lab comparability and reduce outcome variations. Relevant organizations, such as the Society for Experimental Stroke (SFES), National Institutes of Health (NIH), National Stroke Association, American Stroke Association, and International Society for Cerebral Blood Flow and Metabolism (ISCBFM) may take a

leadership role in promoting development of an SOP for stroke models.

### B. TECHNICAL CHALLENGES IN STROKE MODELS

Some variations in stroke model outcomes are due to technical difficulties in stroke model procedures. The following technical challenges comprise a checklist for the improvement of stroke model quality:

* Non-invasive blood pressure monitor with constant reading.

* Minimally invasive remote brain temperature monitoring.

* Warm chamber equipped with PID temperature controller and in vivo feedback.

* Remote/in vivo blood gas analyser, glucose monitor.

* Light weight microclips for MCAO.

* Micromanipulator for applying microclips, bipolar coagulator on MCA.

* Uniformly shaped and lysing-controllable emboli for embolic MCAO model.

* Optimized surgical tools for MCAO models.

* Method for in vivo detection of cerebral arterial structure variation.

## ACKNOWLEDGEMENT

This work was supported by NIH grants 5T32NS051147-02 and NS-21076-24. The authors appreciate and acknowledge Dr. Levine at Mount Sinai School of Medicine for his contribution on revising this paper.

## REFERENCES

* Abraini JH, David HN, Nicole O, MacKenzie ET, Buisson A, Lemaire M. (2004) Neuroprotection by nitrous oxide and xenon and its relation to minimum alveolar concentration. *Anesthesiology* 101:260-261; author reply 261

* Bayona NA, Gelb AW, Jiang Z, Wilson JX, Urquhart BL, Cechetto DF. (2004) Propofol neuroprotection in cerebral ischemia and its effects on low-molecular-weight antioxidants and skilled motor tasks. *Anesthesiology* 100:1151-1159

* Becker K, Kindrick D, Relton J, Harlan J, Winn R. (2001) Antibody to the alpha4 integrin decreases infarct size in transient focal cerebral ischemia in rats. *Stroke* 32:206-211

* Bederson JB, Pitts LH, Germano SM, Nishimura MC, Davis RL, Bartkowski HM. (1986a) Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats. *Stroke* 17:1304-1308

* Bederson JB, Pitts LH, Tsuji M, Nishimura MC, Davis RL, Bartkowski H. (1986b) Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. *Stroke* 17:472-476

* Beech JS, Williams SC, Campbell CA, Bath PM, Parsons AA, Hunter AJ, Menon DK. (2001) Further characterisation of a thromboembolic model of stroke in the rat. *Brain Res* 895:18-24
Belayev L, Alonso OF, Busto R, Zhao W, Ginsberg MD. (1996) Middle cerebral artery occlusion in the rat by intraluminal suture. Neurological and pathological evaluation of an improved model. *Stroke* 27:1616-1622; discussion 1623

Belayev L, Busto R, Zhao W, Fernandez G, Ginsberg MD. (1999) Middle cerebral artery occlusion in the mouse by intraluminal suture coated with poly-L-lysine: neurological and histological validation. *Brain Res* 833:181-190

Benedek A, Moricz K, Juranyi Z, Gigler G, Levay G, Harsing LG, Jr., Matyus P, Szenasi G, Albert M. (2006) Use of TTC staining for the evaluation of tissue injury in the early phases of reperfusion after focal cerebral ischemia in rats. *Brain Res* 1116:159-165

Biernaskie J, Corbett D, Peeling J, Wells J, Lei H. (2001) A serial MR study of cerebral blood flow changes and lesion development following endothelin-1-induced ischemia in rats. *Magn Reson Med* 46:827-830

Bostek CC. (1989) Oxygen toxicity: an introduction. AANA J 57:231-237

Bottiger BW, Teschendorf P, Krumnikl JJ, Vogel P, Galmbacher R, Schmitz B, Motsch J, Martin E, Gass P. (1999) Global cerebral ischemia due to cardiocirculatory arrest in mice causes neuronal degeneration and early induction of transcription factor genes in the hippocampus. *Brain Res Mol Brain Res* 65:135-142

Brint S, Jacewicz M, Kiessling M, Tanabe J, Pulsinelli W. (1988) Focal brain ischemia in the rat: methods for reproducible neocortical infarction using tandem occlusion of the distal middle cerebral and ipsilateral common carotid arteries. *J Cereb Blood Flow Metab* 8:474-485

Buchan AM, Xue D, Slivka A. (1992) A new model of temporary focal neocortical ischemia in the rat. *Stroke* 23:273-279

Buchhold B, Mogoanta L, Suofu Y, Hamm A, Walker L, Kessler C, Popa-Wagner A. (2007) Environmental enrichment improves functional and neuropathological indices following stroke in young and aged rats. *Restor Neurol Neurosci* 25:467-484

Bulte DP, Chiarelli PA, Wise RG, Jezzard P. (2007) Cerebral perfusion response to hyperoxia. *J Cereb Blood Flow Metab* 27:69-75

Busch E, Kruger K, Hossmann KA. (1997) Improved model of thromboembolic stroke and rt-PA induced reperfusion in the rat. *Brain Res* 778:16-24

Candelario-Jalil E, Munoz E, Fiebich BL. (2008) Detrimental effects of tropisetron on permanent ischemic stroke in the rat. *BMC Neurosci* 9:19

Chang S, Jiang X, Zhao C, Lee C, Ferriero DM. (2008) Exogenous low dose hydrogen peroxide increases hypoxia-inducible factor-1alpha protein expression and induces preconditioning protection against ischemia in primary cortical neurons. *Neurosci Lett* 441:134-138

Chen F, Suzuki Y, Nagai N, Peeters R, Sun X, Coudyzer W, Marchal G, Ni Y. (2004) Rat cerebral ischemia induced with photochemical occlusion of proximal middle cerebral artery: a stroke model for MR imaging research. *MAGMA* 17:103-108

Chen J, Li Y, Wang L, Zhang Z, Lu D, Lu M, Chopp M. (2001) Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats. *Stroke* 32:1005-1011

Chen ST, Hsu CY, Hogan EL, Maricq H, Balentine JD. (1986) A model of focal ischemic stroke in the rat: reproducible extensive cortical infarction. *Stroke* 17:738-743

Chen Y, Ito A, Takai K, Saito N. (2008) Blocking pterygopalatine arterial blood flow decreases infarct volume variability in a mouse model of intraluminal suture middle cerebral artery occlusion. *J Neurosci Methods* 174:18-24

Cho S, Szeto HH, Kim E, Kim H, Tolhurst AT, Pinto JT. (2007) A novel cell-permeable antioxidant peptide, SS31, attenuates ischemic brain injury by down-regulating CD36. *J Biol Chem* 282:4634-4642

Coert BA, Anderson RE, Meyer FB. (1999) Reproducibility of cerebral cortical infarction in the wistar rat after middle cerebral artery occlusion. *J Stroke Cerebrovasc Dis* 8:380-387

Coert BA, Anderson RE, Meyer FB. (2003) Is neuroprotective efficacy of nNOS inhibitor 7-NI dependent on ischemic intracellular pH? *Am J Physiol Heart Circ Physiol* 284:H151-159

Colbourne F, Corbett D, Zhao Z, Yang J, Buchan AM. (2000) Prolonged but delayed postischemic hypothermia: a long-term outcome study in the rat middle cerebral artery occlusion model. *J Cereb Blood Flow Metab* 20:1702-1708

Cole DJ, Drummond JC, Shapiro HM, Zornow MH. (1990) Influence of hypotension and hypotensive technique on the area of profound reduction in cerebral blood flow during focal cerebral ischaemia in the rat. *Br J Anaesth* 64:498-502

Czurko A, Nishino H. (1993) 'Collapsed' (argyrophilic, dark) neurons in rat model of transient focal cerebral ischemia. *Neurosci Lett* 162:71-74

David CA, Prado R, Dietrich WD. (1996) Cerebral protection by intermittent reperfusion during temporary focal ischemia in the rat. *J Neurosurg* 85:923-928

David HN, Haelewyn B, Rouillon C, Lecoq M, Chazalviel L, Apiou G, Risso JJ, Lemaire M, Abraini JH. (2008) Neuroprotective effects of xenon: a therapeutic window of opportunity in rats subjected to transient cerebral ischemia. *Faseb J* 22:1275-1286

De Ryck M, Keersmaekers R, Duytschaever H, Claes C, Clincke G, Janssen M, Van Reet G. (1996) Lubeluzole protects sensorimotor function and reduces infarct size in a photochemical stroke model in rats. *J Pharmacol Exp Ther* 279:748-758

De Ryck M, Van Reempts J, Borgers M, Wauquier A, Janssen PA. (1989) Photochemical stroke model: flunarizine prevents sensorimotor deficits after neocortical infarcts in rats. *Stroke* 20:1383-1390

De Ryck M, Verhoye M, Van der Linden AM. (2000) Diffusion-weighted MRI of infarct growth in a rat photochemical stroke model: effect of lubeluzole. *Neuropharmacology* 39:691-702

DeBow S, Colbourne F. (2003) Brain temperature measurement and regulation in awake and freely moving rodents. *Methods* 30:167-171

Dettmers C, Hartmann A, Rommel T, Kramer S, Pappata S, Young A, Hartmann S, Zierz S, MacKenzie ET, Baron JC. (1994) Immersion and perfusion staining with 2,3,5-triphenyltetrazolium chloride (TTC) compared to mitochondrial enzymes 6 hours after MCA-occlusion in primates. *Neurol Res* 16:205-208

Dimitrijevic OB, Stamatovic SM, Keep RF, Andjelkovic AV. (2007) Absence of the chemokine receptor CCR2 protects against cerebral ischemia/reperfusion injury in mice. *Stroke* 38:1345-1353

Diringer MN. (2008) Hyperoxia: good or bad for the injured brain? *Curr Opin Crit Care* 14:167-171

Dirnagl U. (2006) Bench to bedside: the quest for quality in experimental stroke research. *J Cereb Blood Flow Metab* 26:1465-1478

Dittmar MS, Vatankhah B, Fehm NP, Schuierer G, Bogdahn U, Horn M, Schlachetzki F. (2006) Fischer-344 rats are unsuitable for the MCAO filament model due to their cerebrovascular anatomy. *J Neurosci Methods* 156:50-54

Drummond DC, Zignani M, Leroux J. (2000) Current status of pH-sensitive liposomes in drug delivery. *Prog Lipid Res* 39:409-460

Drummond JC, Cole DJ, Patel PM, Reynolds LW. (1995) Focal cerebral ischemia during anesthesia with etomidate, isoflurane, or thiopental: a comparison of the extent of cerebral injury. *Neurosurgery* 37:742-748; discussion 748-749

Eichenbaum JW, Pevsner PH, Pivawer G, Kleinman GM, Chiriboga L, Stern A, Rosenbach A, Iannuzzi K, Miller DC. (2002) A murine photochemical stroke model with histologic correlates of apoptotic and nonapoptotic mechanisms. *J Pharmacol Toxicol Methods* 47:67-71
Erdem AF, Cesur M, Alici HA, Erdogan F, Dogan N, Kursad H, Yuksek MS. (2005) Effects of sevoflurane and desflurane in CA1 after incomplete cerebral ischemia in rats. *Saudi Med J* 26:1424-1428

Erdo F, Berzsenyi P, Nemet L, Andrasi F. (2006) Talampanel improves the functional deficit after transient focal cerebral ischemia in rats. A 30-day follow up study. *Brain Res Bull* 68:269-276

Estevez AY, Phillis JW. (1997) The phospholipase A2 inhibitor, quinacrine, reduces infarct size in rats after transient middle cerebral artery occlusion. *Brain Res* 752:203-208

Fisher M. (2003) Recommendations for Advancing Development of Acute Stroke Therapies: Stroke Therapy Academic Industry Roundtable 3. *Stroke* 34:1539-1546

Fisher M, (Stroke Therapy Academic Industry Roundtable, I. V.). (2005) Enhancing the Development and Approval of Acute Stroke Therapies: Stroke Therapy Academic Industry Roundtable. *Stroke* 36:1808-1813

Fisher M, Feuerstein G, Howells DW, Hurn PD, Kent TA, Savitz SI, Lo EH. (2009) Update of the stroke therapy academic industry roundtable preclinical recommendations. *Stroke* 40:2244-2250

Fisher M, Hanley DF, Howard G, Jauch EC, Warach S, for the SG. (2007) Recommendations From the STAIR V Meeting on Acute Stroke Trials, Technology and Outcomes. *Stroke* 38:245-248

Florian B, Vintilescu R, Balseanu AT, Buga AM, Grisk O, Walker LC, Kessler C, Popa-Wagner A. (2008) Long-term hypothermia reduces infarct volume in aged rats after focal ischemia. *Neurosci Lett* 438:180-185

Futrell N, Watson BD, Dietrich WD, Prado R, Millikan C, Ginsberg MD. (1988) A new model of embolic stroke produced by photochemical injury to the carotid artery in the rat. *Ann Neurol* 23:251-257

Garcia JH, Yoshida Y, Chen H, Li Y, Zhang ZG, Lian J, Chen S, Chopp M. (1993) Progression from ischemic injury to infarct following middle cerebral artery occlusion in the rat. *Am J Pathol* 142:623-635

Gaudinski MR, Henning EC, Miracle A, Luby M, Warach S, Latour LL. (2008) Establishing final infarct volume: stroke lesion evolution past 30 days is insignificant. *Stroke* 39:2765-2768

Goldlust EJ, Paczynski RP, He YY, Hsu CY, Goldberg MP. (1996) Automated measurement of infarct size with scanned images of triphenyltetrazolium chloride-stained rat brains. *Stroke* 27:1657-1662

Grasshoff C, Rudolph U, Antkowiak B. (2005) Molecular and systemic mechanisms of general anaesthesia: the 'multi-site and multiple mechanisms' concept. *Curr Opin Anaesthesiol* 18:386-391

Greco R, Amantea D, Blandini F, Nappi G, Bagetta G, Corasaniti MT, Tassorelli C. (2007) Neuroprotective effect of nitroglycerrin in a rodent model of ischemic stroke: evaluation of Bcl-2 expression. *Int Rev Neurobiol* 82:423-435

Greene SA, Thurmon JC, Tranquilli WJ, Benson GJ. (1987) Effect of yohimbine on xylazine-induced hypoinsulinemia and hyperglycemia in mares. *Am J Vet Res* 48:676-678

Haelewyn B, David HN, Rouillon C, Chazalviel L, Lecocq M, Risse JJ, Lemaire M, Abraini JH. (2008) Neuroprotection by nitrous oxide: facts and evidence. *Crit Care Med* 36:2651-2659

Haelewyn B, Yvon A, Hanouz JL, MacKenzie ET, Ducouret P, Gerard JL, Roussel S. (2003) Desflurane affords greater protection than halothane against focal cerebral ischaemia in the rat. *Br J Anaesth* 91:390-396

Harada T, Kano T, Katayama Y, Matsuzaki T, Tejima E, Koshinaga M. (2005) Tissue plasminogen activator extravasated through the cerebral vessels: evaluation using a rat thromboembolic stroke model. *Thromb Haemost* 94:791-796

Horie N, Maag AL, Hamilton SA, Shichinohe H, Bliss TM, Steinberg GK. (2008) Mouse model of focal cerebral ischemia using endothelin-1. *J Neurosci Methods* 173:286-290

Horita Y, Honmou O, Harada K, Houkin K, Hamada H, Kocsis JD. (2006) Intravenous administration of glial cell line-derived neurotrophic factor gene-modified human mesenchymal stem cells protects against injury in a cerebral ischemia model in the adult rat. *J Neurosci Res* 84:1495-1504

Huang J, Kim LJ, Poisik A, Pinsky DJ, Connolly ES, Jr. (1998) Does poly-L-lysine coating of the middle cerebral artery occlusion suture improve infarct consistency in a murine model? *J Stroke Cerebrovasc Dis* 7:296-301

Hungerhuber E, Zausinger S, Westermaier T, Plesnila N, Schmid-Elsaesser R. (2006) Simultaneous bilateral laser Doppler fluxmetry and electrophysiological recording during middle cerebral artery occlusion in rats. *J Neurosci Methods* 154:109-115

Isayama K, Pitts LH, Nishimura MC. (1991) Evaluation of 2,3,5-triphenyltetrazolium chloride staining to delineate rat brain infarcts. *Stroke* 22:1394-1398

Iwatsuki N, Takahashi M, Ono K, Tajima T. (1994) Hyperbaric oxygen combined with nicardipine administration accelerates neurologic recovery after cerebral ischemia in a canine model. *Crit Care Med* 22:858-863

Jia X, Koenig MA, Shin HC, Zhen G, Yamashita S, Thakor NV, Geocadin RG. (2006) Quantitative EEG and neurological recovery with therapeutic hypothermia after asphyxial cardiac arrest in rats. *Brain Res* 1111:166-175

Joshi CN, Jain SK, Murthy PS. (2004) An optimized triphenyltetrazolium chloride method for identification of cerebral infarcts. *Brain Res Brain Res Protoc* 13:11-17

Kano T, Harada T, Katayama Y. (2003) Infiltration of tissue plasminogen activator through cerebral vessels: evaluation using a rat thromboembolic stroke model. *Acta Neurochir Suppl* 86:167-168

Kano T, Harada T, Katayama Y. (2005) Attenuation of extravasation of tissue plasminogen activator by the free radical scavenger, edaravone: evaluation in a rat thromboembolic stroke model. *Neurol Res* 27:499-502

Kawaguchi M, Drummond JC, Cole DJ, Kelly PJ, Spurlock MP, Patel PM. (2004) Effect of isoflurane on neuronal apoptosis in rats subjected to focal cerebral ischemia. *Anesth Analg* 98:798-805, table of contents

Khan M, Jatana M, Elango C, Paintlia AS, Singh AK, Singh I. (2006) Cerebrovascular protection by various nitric oxide donors in rats after experimental stroke. *Nitric Oxide* 15:114-124

Kilic E, Hermann DM, Hossmann KA. (1998) A reproducible model of thromboembolic stroke in mice. *Neuroreport* 9:2967-2970

Kim E, Tolhurst AT, Qin LY, Chen XY, Febbraio M, Cho S. (2008) CD36/fatty acid translocase, an inflammatory mediator, is involved in hyperlipidemia-induced exacerbation in ischemic brain injury. *J Neurosci* 28:4661-4670

Kim Y, Busto R, Dietrich WD, Kraydieh S, Ginsberg MD. (1996) Delayed postischemic hyperthermia in awake rats worsens the histopathological outcome of transient focal cerebral ischemia. *Stroke* 27:2274-2280; discussion 2281

Kitagawa K, Matsumoto M, Hori M. (2004) Cerebral ischemia in 5-lipoxygenase knockout mice. *Brain Res* 1004:198-202

Kleinschnitz C, Pozgajova M, Pham M, Bendszus M, Nieswandt B, Stoll G. (2007) Targeting platelets in acute experimental stroke: impact of glycoprotein Ib, VI, and IIb/IIIa blockade on infarct size, functional outcome, and intracranial bleeding. *Circulation* 115:2323-2330

Koizumi J, Yoshida Y, Nakazawa T, Ooneda G. (1986) Experimental studies of ischemic brain edema, I: a new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area. *Jpn J Stroke* 8:1-8

Kontos HA, Raper AJ, Patterson JL. (1977a) Analysis of vasoactivity of local pH, PCO2 and bicarbonate on pial vessels. *Stroke* 8:358-360

Kontos HA, Wei EP, Raper AJ, Patterson JL, Jr. (1977b) Local mechanism of CO2 action of cat pial arterioles. *Stroke* 8:226-229
Krueger K, Busch E. (2002) Protocol of a thromboembolic stroke model in the rat: review of the experimental procedure and comparison of models. *Invest Radiol* 37:600-608

Kudo M, Aoyama A, Ichimori S, Fukunaga N. (1982) An animal model of cerebral infarction. Homologous blood clot emboli in rats. *Stroke* 13:505-508

Leach MJ, Swan JH, Eisenthal D, Dopson M, Nobbs M. (1993) BW619C89, a glutamate release inhibitor, protects against focal cerebral ischemic damage. *Stroke* 24:1063-1067

Lee JK, Kim JE, Sivula M, Strittmatter SM. (2004) Nogo receptor antagonism promotes stroke recovery by enhancing axonal plasticity. *J Neurosci* 24:6209-6217

Lin TN, He YY, Wu G, Khan M, Hsu CY. (1993) Effect of brain edema on infarct volume in a focal cerebral ischemia model in rats. *Stroke* 24:117-121

Lindner MD, Gribkoff VK, Donlan NA, Jones TA. (2003) Long-lasting functional disabilities in middle-aged rats with small cerebral infarcts. *J Neurosci* 23:10913-10922

Liszczak TM, Hedley-Whyte ET, Adams JF, Han DH, Kolluri VS, Vacanti FX, Heros RC, Zervas NT. (1984) Limitations of trazolium salts in delineating infarcted brain. *Acta Neuropathol* 65:150-157

Liu S, Guo Y. (2000a) [Dynamic penumbra in a rat model of focal cerebral ischemia and reperfusion]. *Zhongguo Yi Xue Ke Xue Yuan Xue Bao* 22:177-181

Liu S, Guo Y. (2000b) [Identification of early irreversible damage area in a rat model of cerebral ischemia and reperfusion]. *Zhongguo Yi Xue Ke Xue Yuan Xue Bao* 22:25-29

Liu S, Levine SR. (2008) The continued promise of neuroprotection for acute stroke treatment. *J Exp Stroke & Transl Med* 1:1-8

Liu S, Liu W, Ding W, Miyake M, Rosenberg GA, Liu KJ. (2006) Electron paramagnetic resonance-guided normobaric hyperoxia treatment protects the brain by maintaining penumbral oxygenation in a rat model of transient focal cerebral ischemia. *J Cereb Blood Flow Metab* 26:1274-1284

Liu S, Shi H, Liu W, Furuichi T, Timmins GS, Liu KJ. (2004) Intersitial pO2 in ischemic penumbra and core are differentially affected following transient focal cerebral ischemia in rats. *J Cereb Blood Flow Metab* 24:343-349

Longa EZ, Weinstein PR, Carlson S, Cummins R. (1989) Reversible middle cerebral artery occlusion without craniectomy in rats. *Stroke* 20:84-91

Lovblad KO, El-Koussy M, Oswald H, Baird AE, Schroth G, Mattle H. (2003) Magnetic resonance imaging of the ischaemic penumbra. *Swiss Med Wkly* 133:551-559

Lozano JD, Abulafia DP, Danton GH, Watson BD, Dietrich WD. (2007) Characterization of a thromboembolic photochemical model of repeated stroke in mice. *J Neurosci Methods* 162:244-254

Lu A, Kurosawa Y, Luskey K, Pyne-Geithman G, Caudell D, Clark J. (2009) Hemorrhagic profile of the fibrinolytic alfimeprase after ischemia and reperfusion. *Neurol Res* 31:209-214

Lundy EF, Solik BS, Frank RS, Lacy PS, Combs DJ, Zelenock GB, D'Alecy LG. (1986) Morphometric evaluation of brain infarcts in rats and gerbils. *J Pharmacol Methods* 16:201-214

Macleod MR, Fisher M, O'Collins V, Sena ES, Dirnagl U, Bath PM, Buchan A, van der Worp HB, Traystman R, Minematsu K, Donnan GA, Howells DW. (2009a) Good laboratory practice: preventing introduction of bias at the bench. *Stroke* 40:e50-52

Macleod MR, Fisher M, O'Collins V, Sena ES, Dirnagl U, Bath PM, Buchan A, van der Worp HB, Traystman RJ, Minematsu K, Donnan GA, Howells DW. (2009b) Reprint: Good laboratory practice: preventing introduction of bias at the bench. *Int J Stroke* 4:3-5

Macleod MR, Fisher M, O'Collins V, Sena ES, Dirnagl U, Bath PM, Buchan A, van der Worp HB, Traystman RJ, Minematsu K, Donnan GA, Howells DW. (2009c) Reprint: Good laboratory practice: preventing introduction of bias at the bench. *J Cereb Blood Flow Metab* 29:221-223

Maggi CA, Meli A. (1986) Suitability of urethane anesthesia for physiopharmacological investigations in various systems. Part 1: General considerations. *Experientia* 42:109-114

Margaill I, Parmentier S, Callebert J, Allix M, Boulu RG, Plotkine M. (1996) Short therapeutic window for MK-801 in transient focal cerebral ischemia in normotensive rats. *J Cereb Blood Flow Metab* 16:107-113

Marion DW. (2004) Controlled normothermia in neurologic intensive care. *Crit Care Med* 32:S43-45

Masada T, Hua Y, Xi G, Ennis SR, Keep RF. (2001) Attenuation of ischemic brain edema and cerebrovascular injury after ischemic preconditioning in the rat. *J Cereb Blood Flow Metab* 21:22-33

Matsushima K, Hakim AM. (1995) Transient forebrain ischemia protects against subsequent focal cerebral ischemia without changing cerebral perfusion. *Stroke* 26:1047-1052

Maysami S, Lan JQ, Minami M, Simon RP. (2008) Proliferating progenitor cells: a required cellular element for induction of ischemic tolerance in the brain. *J Cereb Blood Flow Metab* 28:1104-1113

McIlvoy L. (2004) Comparison of brain temperature to core temperature: a review of the literature. *J Neurosci Nurs* 36:23-31

Meden P, Andersen M, Overgaard K, Rasmussen RS, Boysen G. (2002) The effects of early insulin treatment combined with thrombolysis in rat embolic stroke. *Neurol Res* 24:399-404

Menzel M, Rieger A, Roth S, Soukup J, Furka I, Miko I, Molnar P, Peuse C, Hennig C, Radke J. (1998) Comparison between continuous brain tissue pO2, pCO2, pH, and temperature and simultaneous cerebrovenous measurement using a multisensor probe in a porcine intracranial pressure model. *J Neurotrauma* 15:265-276

Miyazawa T, Tamura A, Fukui S, Hossmann KA. (2003) Effect of mild hypothermia on focal cerebral ischemia. Review of experimental studies. *Neurol Res* 25:457-464

Morikawa E, Ginsberg MD, Dietrich WD, Duncan RC, Kraydieh S, Globus MY, Busto R. (1992) The significance of brain temperature in focal cerebral ischemia: histopathological consequences of middle cerebral artery occlusion in the rat. *J Cereb Blood Flow Metab* 12:380-389

Nakajima Y, Moriwaki G, Ikeda K, Fujise Y. (1997) The effects of sevoflurane on recovery of brain energy metabolism after cerebral ischemia in the rat: a comparison with isoflurane and halothane. *Anesth Analg* 85:593-599

Nikolova S, Moyanova S, Hughes S, Bellyou-Camilleri M, Lee TY, Bartha R. (2009) Endothelin-1 induced MCAO: dose dependency of cerebral blood flow. *J Neurosci Methods* 179:22-28

Noor R, Wang CX, Shuaib A. (2003) Effects of hyperthermia on infarct volume in focal embolic model of cerebral ischemia in rats. *Neurosci Lett* 349:130-132

Noor R, Wang CX, Shuaib A. (2005) Hyperthermia masks the neuroprotective effects of tissue plaminogen activator. *Stroke* 36:665-669

Nussmeier NA. (2005) Management of temperature during and after cardiac surgery. *Tex Heart Inst J* 32:472-476

Ohta H, Terao Y, Shintani Y, Kiyota Y. (2007) Therapeutic time window of post-ischemic mild hypothermia and the gene expression associated with the neuroprotection in rat focal cerebral ischemia. *Neurosci Res* 57:424-433

Oliff HS, Weber E, Eilon G, Marek P. (1995a) The role of strain/vendor differences on the outcome of focal ischemia induced by intraluminal middle cerebral artery occlusion in the rat. *Brain Res* 675:20-26

Oliff HS, Weber E, Miyazaki B, Marek P. (1995b) Infarct volume varies with rat strain and vendor in focal cerebral ischemia induced by transcranial middle cerebral artery occlusion. *Brain Res* 699:329-331

Olsson T, Wieloch T, Smith ML. (2003) Brain damage in a mouse model of global cerebral ischemia. Effect of NMDA receptor blockade. *Brain Res* 982:260-269
Onal MZ, Li F, Tatlisumak T, Locke KW, Sandage BW, Jr., Fisher M. (1997) Synergistic effects of citicoline and MK-801 in temporary experimental focal ischemia in rats. *Stroke* 28:1060-1065

Orset C, Macrez R, Young AR, Panthou D, Angles-Cano E, Maubert E, Agin V, Vivien D. (2007) Mouse model of in situ thromboembolic stroke and reperfusion. *Stroke* 38:2771-2778

Ostrovskaya RU, Romanova GA, Barskov IV, Shanina EV, Gudasheva TA, Victorov IV, Voronina TA, Seredenin SB. (1999) Memory restoring and neuroprotective effects of the proline-containing dipeptide, GVS-111, in a photochemical stroke model. *Behav Pharmacol* 10:549-553

Overgaard K, Meden P. (2000) Influence of different fixation procedures on the quantification of infarction and oedema in a rat model of stroke. *Neuropathol Appl Neurobiol* 26:243-250

Parsons MW, Barber PA, Desmond PM, Baird TA, Darby DG, Byrnes G, Tress BM, Davis SM. (2002) Acute hyperglycemia adversely affects stroke outcome: a magnetic resonance imaging and spectroscopy study. *Ann Neurol* 52:20-28

Payne RS, Akca O, Roewer N, Schurr A, Kehl F. (2005) Sevoflurane-induced preconditioning protects against cerebral ischemic neuronal damage in rats. *Brain Res* 1034:147-152

Pettigrew LC, Holtz ML, Craddock SD, Minger SL, Hall N, Geddes JW. (1996) Microtubular proteolysis in focal cerebral ischemia. *J Cereb Blood Flow Metab* 16:1189-1202

Pignataro G, Simon RP, Boison D. (2007a) Transgenic overexpression of adenosine kinase aggravates cell death in ischemia. *J Cereb Blood Flow Metab* 27:1-5

Pignataro G, Simon RP, Xiong ZG. (2007b) Prolonged activation of ASIC1a and the time window for neuroprotection in cerebral ischaemia. *Brain* 130:151-158

Pignataro G, Studer FE, Wilz A, Simon RP, Boison D. (2007c) Neuroprotection in ischemic mouse brain induced by stem cell-derived brain implants. *J Cereb Blood Flow Metab* 27:919-927

Proescholdt M, Heimann A, Kempski O. (2001) Neuroprotection of S(+) ketamine isomer in global forebrain ischemia. *Brain Res* 904:245-251

Rajkumar K, Barron D, Lewitt MS, Murphy LJ. (1995) Growth retardation and hyperglycemia in insulin-like growth factor binding protein-1 transgenic mice. *Endocrinology* 136:4029-4034

Rajkumar K, Dheen ST, Murphy LJ. (1996) Hyperglycemia and impaired glucose tolerance in IGF binding protein-1 transgenic mice. *Am J Physiol* 270:E565-571

Reglodi D, Tamas A, Lengvari I. (2003) Examination of sensorimotor performance following middle cerebral artery occlusion in rats. *Brain Res Bull* 59:459-466

Renzulli P, Laffer UT. (2005) Learning curve: the surgeon as a prognostic factor in colorectal cancer surgery. *Recent Results Cancer Res* 165:86-104

Rogers DC, Campbell CA, Stretton JL, Mackay KB. (1997) Correlation between motor impairment and infarct volume after permanent and transient middle cerebral artery occlusion in the rat. *Stroke* 28:2060-2065; discussion 2066

Romanos E, Planas AM, Amaro S, Chamorro A. (2007) Uric acid reduces brain damage and improves the benefits of rt-PA in a rat model of thromboembolic stroke. *J Cereb Blood Flow Metab* 27:14-20

Roof RL, Schielke GP, Ren X, Hall ED. (2001) A comparison of long-term functional outcome after 2 middle cerebral artery occlusion models in rats. *Stroke* 32:2648-2657

Rordorf G, Cramer SC, Efird JT, Schwamm LH, Buonanno F, Koroshetz WJ. (1997) Pharmacological elevation of blood pressure in acute stroke. Clinical effects and safety. *Stroke* 28:2133-2138

Rusyniak DE, Kirk MA, May JD, Kao LW, Brizendine EJ, Welch JL, Cordell WH, Alonso RJ. (2003) Hyperbaric oxygen therapy in acute ischemic stroke: results of the Hyperbaric Oxygen in Acute Ischemic Stroke Trial Pilot Study. *Stroke* 34:571-574

Saha JK, Xia J, Grondin JM, Engle SK, Jakubowski JA. (2005) Acute hyperglycemia induced by ketamine/xylazine anesthesia in rats: mechanisms and implications for preclinical models. *Exp Biol Med (Maywood)* 230:777-784

Saver JL, Albers GW, Dunn B, Johnston KC, Fisher M. (2009) Stroke Therapy Academic Industry Roundtable (STAIR) recommendations for extended window acute stroke therapy trials. *Stroke* 40:2594-2600

Savitz SL. (2007) A critical appraisal of the NXY-059 neuroprotection studies for acute stroke: a need for more rigorous testing of neuroprotective agents in animal models of stroke. *Exp Neurol* 205:20-25

Sceniak MP, Maciver MB. (2006) Cellular actions of urethane on rat visual cortical neurons in vitro. *J Neurophysiol* 95:3865-3874

Schallert T. (2006) Behavioral tests for preclinical intervention assessment. *NeuroRx* 3:497-504

Schielke GP, Kupina NC, Boxer PA, Bigge CF, Welty DF, Iadecola C. (1999) The neuroprotective effect of the novel AMPA receptor antagonist PD152247 (PNQX) in temporary focal ischemia in the rat. *Stroke* 30:1472-1477

Schmid-Elsaesser R, Zausinger S, Hungerhuber E, Baethmann A, Reulen HJ. (1998) A critical reevaluation of the intraluminal thread model of focal cerebral ischemia: evidence of inadvertent premature reperfusion and subarachnoid hemorrhage in rats by laser-Doppler flowmetry. *Stroke* 29:2162-2170

Selman WR, Bhatti SU, Rosenberg CC, Lust WD, Ratcheson RA. (1994) Temporary vessel occlusion in spontaneously hypertensive and normotensive rats. Effect of single and multiple episodes on tissue metabolism and volume of infarction. *J Neurosurg* 80:1085-1090

Shah ZA, Namiranian K, Klaus J, Kibler K, Dore S. (2006) Use of an optimized transient occlusion of the middle cerebral artery protocol for the mouse stroke model. *J Stroke Cerebrovasc Dis* 15:133-138

Sheng H, Laskowitz DT, Pearlstein RD, Warner DS. (1999) Characterization of a recovery global cerebral ischemia model in the mouse. *J Neurosci Methods* 88:103-109

Shimamura N, Matchett G, Tsubokawa T, Ohkuma H, Zhang J. (2006a) Comparison of silicon-coated nylon suture to plain nylon suture in the rat middle cerebral artery occlusion model. *J Neurosci Methods* 156:161-165

Shimamura N, Matchett G, Yatsushige H, Calvert JW, Ohkuma H, Zhang J. (2006b) Inhibition of integrin alphavbeta3 ameliorates focal cerebral ischemic damage in the rat middle cerebral artery occlusion model. *Stroke* 37:1902-1909

Simard JM, Yurovsky V, Tsymbalyuk N, Melnichenko L, Ivanova S, Gerzanich V. (2009) Protective effect of delayed treatment with low-dose glibenclamide in three models of ischemic stroke. *Stroke* 40:604-609

Singhal AB. (2007) A review of oxygen therapy in ischemic stroke. *Neurol Res* 29:173-183

Singhal AB, Benner T, Roccatagliata L, Koroshetz WJ, Schaefer PW, Lo EH, Buonanno FS, Gonzalez RG, Sorensen AG. (2005) A pilot study of normobaric oxygen therapy in acute ischemic stroke. *Stroke* 36:797-802

Singhal AB, Wang X, Sumii T, Mori T, Lo EH. (2002) Effects of normobaric hyperoxia in a rat model of focal cerebral ischemia-reperfusion. *J Cereb Blood Flow Metab* 22:861-868

Siniscalchi A, Zona C, Guatteo E, Mercuri NB, Bernardi G. (1998) An electrophysiological analysis of the protective effects of felbamate, lamotrigine, and lidocaine on the functional recovery from in vitro ischemia in rat neocortical slices. *Synapse* 30:371-379

Solaroglu I, Tsubokawa T, Cahill J, Zhang JH. (2006) Anti-apoptotic effect of granulocyte-colony stimulating factor after focal cerebral ischemia in the rat. *Neuroscience* 143:965-974

Spratt NJ, Fernandez J, Chen M, Rewell S, Cox S, van Raay L, Hogan L, Howells DW. (2006) Modification of the method of thread manufacture improves stroke induction rate and re-
duces mortality after thread-occlusion of the middle cerebral artery in young or aged rats. *J Neurosci Methods* 155:285-290

STAIR Group. (1999) Recommendations for Standards Regarding Preclinical Neuroprotective and Restorative Drug Development. *Stroke* 30:2752-2758

STAIR Group. (2001) Recommendations for Clinical Trial Evaluation of Acute Stroke Therapies. *Stroke* 32:1598-1606

Stewart VC, Land JM, Clark JB, Heales SJ. (1998) Comparison of mitochondrial respiratory chain enzyme activities in rodent astrocytes and neurones and a human astrocytoma cell line. *Neurosci Lett* 247:201-203

Stroke Therapy Academic Industry Roundtable. (1999) Recommendations for Standards Regarding Preclinical Neuroprotective and Restorative Drug Development. *Stroke* 30:2752-2758

Swanson RA, Morton MT, Tsao-Wu G, Savalos RA, Davidson C, Sharp FR. (1990) A semiautomated method for measuring brain infarct volume. *J Cereb Blood Flow Metab* 10:290-293

Takahashi M, Iwatsuki N, Ono K, Tajima T, Akama M, Koga Y. (1992) Hyperbaric oxygen therapy accelerates neurologic recovery after 15-minute complete global cerebral ischemia in dogs. *Crit Care Med* 20:1588-1594

Tamura A, Graham DI, McCulloch J, Teasdale GM. (1981a) Focal cerebral ischaemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion. *J Cereb Blood Flow Metab* 1:53-60

Tamura A, Graham DI, McCulloch J, Teasdale GM. (1981b) Focal cerebral ischaemia in the rat: 2. Regional cerebral blood flow determined by [14C]iodoantipyrine autoradiography following middle cerebral artery occlusion. *J Cereb Blood Flow Metab* 1:61-69

Tang J, Liu J, Zhou C, Ostanin D, Grisham MB, Neil Granger D, Zhang JH. (2005) Role of NADPH oxidase in the brain injury of intracerebral hemorrhage. *J Neurochem* 94:1342-1350

Tejima E, Katayama Y, Suzuki Y, Kano T, Lo EH. (2001) Hemorrhagic transformation after fibrinolysis with tissue plasminogen activator: evaluation of role of hypertension with rat thromboembolic stroke model. *Stroke* 32:1336-1340

Thurmon JC, Steffey EP, Zinkl JG, Woliner M, Howland D, Jr. (1984) Xylazine causes transient dose-related hyperglycemia and increased urine volumes in mares. *Am J Vet Res* 45:224-227

Toomey JR, Valocik RE, Koster PF, Gabriel MA, McVey M, Hart TK, Ohlstein EH, Parsons AA, Barone FC. (2002) Inhibition of factor IX(a) is protective in a rat model of thromboembolic stroke. *Stroke* 33:578-585

Tsai SK, Lin SM, Hung WC, Mok MS, Chih CL, Huang SS. (2004) The effect of desflurane on ameliorating cerebral infarction in rats subjected to focal cerebral ischemia-reperfusion injury. *Life Sci* 74:2541-2549

Tsubokawa T, Jadhav V, Solaroglu I, Shiokawa Y, Konishi Y, Zhang JH. (2007) Lecithinized superoxide dismutase improves outcomes and attenuates focal cerebral ischemic injury via antiapoptotic mechanisms in rats. *Stroke* 38:1057-1062

Tsubokawa T, Solaroglu I, Yatsushige H, Cahill J, Yata K, Zhang JH. (2006a) Cathepsin and calpain inhibitor E64d attenuates matrix metalloproteinase-9 activity after focal cerebral ischemia in rats. *Stroke* 37:1888-1894

Tsubokawa T, Yamaguchi-Okada M, Calvert JW, Solaroglu I, Shimamura N, Yata K, Zhang JH. (2006b) Neurovascular and neuronal protection by E64d after focal cerebral ischemia in rats. *J Neurosci Res* 84:832-840

Tsuchiya D, Hong S, Kayama T, Panter SS, Weinstein PR. (2003) Effect of suture size and carotid clip application upon blood flow and infarct volume after permanent and temporary middle cerebral artery occlusion in mice. *Brain Res* 970:131-139

Tureyen K, Vemuganti R, Sailor KA, Dempsey RJ. (2004) Infarct volume quantification in mouse focal cerebral ischemia: a

comparison of triphenyltetrazolium chloride and cresyl violet staining techniques. *J Neurosci Methods* 139:203-207

van der Worp HB, de Haan P, Morrema E, Kalkman CJ. (2005) Methodological quality of animal studies on neuroprotection in focal cerebral ischaemia. *J Neurol* 252:1108-1114

Velisek L. (1998) Extracellular acidosis and high levels of carbon dioxide suppress synaptic transmission and prevent the induction of long-term potentiation in the CA1 region of rat hippocampal slices. *Hippocampus* 8:24-32

Vornov JJ, Thomas AG, Jo D. (1996) Protective effects of extracellular acidosis and blockade of sodium/hydrogen ion exchange during recovery from metabolic inhibition in neuronal tissue culture. *J Neurochem* 67:2379-2389

Vosko MR, Busch E, Burggraf D, Bultemeier G, Hamann GF. (2003) Microvascular basal lamina damage in thromboembolic stroke in a rat model. *Neurosci Lett* 353:217-220

Wallsh E, Weinstein GS, Franzone A, Clavel A, Rossi PA, Kreps E. (1986) Inflammation of the coronary arteries in patients with unstable angina. *Tex Heart Inst J* 13:105-108

Wang CX, Yang T, Shuaib A. (2001) An improved version of embolic model of brain ischemic injury in the rat. *J Neurosci Methods* 109:147-151

Warner DS, Takaoka S, Wu B, Ludwig PS, Pearlstein RD, Brinkhous AD, Dexter F. (1996) Electroencephalographic burst suppression is not required to elicit maximal neuroprotection from pentobarbital in a rat model of focal cerebral ischemia. *Anesthesiology* 84:1475-1484

Weber ML, Taylor CP. (1994) Damage from oxygen and glucose deprivation in hippocampal slices is prevented by tetrodotoxin, lidocaine and phenytoin without blockade of action potentials. *Brain Res* 664:167-177

Wetzel M, Li L, Harms KM, Roitbak T, Ventura PB, Rosenberg GA, Khokha R, Cunningham LA. (2008) Tissue inhibitor of metalloproteinases-3 facilitates Fas-mediated neuronal cell death following mild ischemia. *Cell Death Differ* 15:143-151

Windle V, Szymanska A, Granter-Button S, White C, Buist R, Peeling J, Corbett D. (2006) An analysis of four different methods of producing focal cerebral ischemia with endothelin-1 in the rat. *Exp Neurol* 201:324-334

Winn HR, Rubio R, Berne RM. (1981) Brain adenosine concentration during hypoxia in rats. *Am J Physiol* 241:H235-242

Wise GR. (1970) Vasopressor-drug therapy for complications of cerebral arteriography. *N Engl J Med* 282:610-612

Woitzik J, Schneider UC, Thome C, Schroeck H, Schilling L. (2006) Comparison of different intravascular thread occlusion models for experimental stroke in rats. *J Neurosci Methods* 151:224-231

Xi GM, Wang HQ, He GH, Huang CF, Wei GY. (2004) Evaluation of murine models of permanent focal cerebral ischemia. *Chin Med J (Engl)* 117:389-394

Xiong L, Zheng Y, Wu M, Hou L, Zhu Z, Zhang X, Lu Z. (2003) Preconditioning with isoflurane produces dose-dependent neuroprotection via activation of adenosine triphosphate-regulated potassium channels after focal cerebral ischemia in rats. *Anesth Analg* 96:233-237, table of contents

Yamamoto S, Tanaka E, Shoji Y, Kudo Y, Inokuchi H, Higashi H. (1997) Factors that reverse the persistent depolarization produced by deprivation of oxygen and glucose in rat hippocampal CA1 neurons in vitro. *J Neurophysiol* 78:903-911

Yamashita T, Ninomiya M, Hernandez Acosta P, Garcia-Verdugo JM, Sunabori T, Sakaguchi M, Adachi K, Kojima T, Hirota Y, Kawase T, Araki N, Abe K, Okano H, Sawamoto K. (2006) Subventricular zone-derived neuroblasts migrate and differentiate into mature neurons in the post-stroke adult striatum. *J Neurosci* 26:6627-6636

Yamauchi A, Shuto H, Dohgu S, Nakano Y, Egawa T, Kataoka Y. (2005) Cyclosporin A aggravates electroshock-induced convulsions in mice with a transient middle cerebral artery occlusion. *Cell Mol Neurobiol* 25:923-928
Yanamoto H, Nagata I, Hashimoto N, Kikuchi H. (1998) Three-vessel occlusion using a micro-clip for the proximal left middle cerebral artery produces a reliable neocortical infarct in rats. *Brain Res Brain Res Protoc* 3:209-220

Yanamoto H, Nagata I, Niitsu Y, Xue JH, Zhang Z, Kikuchi H. (2003) Evaluation of MCAO stroke models in normotensive rats: standardized neocortical infarction by the 3VO technique. *Exp Neurol* 182:261-274

Yang G, Kitagawa K, Matsushita K, Mabuchi T, Yagita Y, Yanagihara T, Matsumoto M. (1997) C57BL/6 strain is most susceptible to cerebral ischemia following bilateral common carotid occlusion among seven mouse strains: selective neuronal death in the murine transient forebrain ischemia. *Brain Res* 752:209-218

Yang Y, Li Q, Miyashita H, Yang T, Shuaib A. (2001) Different dynamic patterns of extracellular glutamate release in rat hippocampus after permanent or 30-min transient cerebral ischemia and histological correlation. *Neuropathology* 21:181-187

Yin D, Zhang JH. (2005) Delayed and multiple hyperbaric oxygen treatments expand therapeutic window in rat focal cerebral ischemic model. *Neurocrit Care* 2:206-211

Yonekura I, Kawahara N, Nakatomi H, Furuya K, Kirino T. (2004) A model of global cerebral ischemia in C57 BL/6 mice. *J Cereb Blood Flow Metab* 24:151-158

Zaremba J. (2004) Hyperthermia in ischemic stroke. *Med Sci Monit* 10:RA148-153

Zarow GJ, Karibe H, States BA, Graham SH, Weinstein PR. (1997) Endovascular suture occlusion of the middle cerebral artery in rats: effect of suture insertion distance on cerebral blood flow, infarct distribution and infarct volume. *Neurol Res* 19:409-416

Zausinger S, Hungerhuber E, Baethmann A, Reulen H, Schmid-Elsaesser R. (2000) Neurological impairment in rats after transient middle cerebral artery occlusion: a comparative study under various treatment paradigms. *Brain Res* 863:94-105

Zhang RL, Chopp M, Zhang ZG, Jiang Q, Ewing JR. (1997a) A rat model of focal embolic cerebral ischemia. *Brain Res* 766:83-92

Zhang Z, Zhang RL, Jiang Q, Raman SB, Cantwell L, Chopp M. (1997b) A new rat model of thrombotic focal cerebral ischemia. *J Cereb Blood Flow Metab* 17:123-135

Zhu CZ, Auer RN. (1995) Graded hypotension and MCA occlusion duration: effect in transient focal ischemia. *J Cereb Blood Flow Metab* 15:980-988

Zlotnik A, Gruenbaum SE, Artru AA, Leibovitz A, Shapira Y. (2008) The Position of Arterial Line Significantly Influences Neurological Assessment in Rats. *ASA Annual Meeting Abstract* 109:A486