Permanent Middle Cerebral Artery Occlusion in Sheep: A Novel Large Animal Model of Focal Cerebral Ischemia

Abstract

As effective stroke treatment by thrombolysis is bound to a narrow time window excluding most patients, numerous experimental treatment strategies have been developed to gain new options for stroke treatment. However, all approaches using neuroprotective agents that have been successfully evaluated in rodents have subsequently failed in clinical trials. Existing large animal models are of significant scientific value, but sometimes limited by ethical drawbacks and mostly do not allow for long-term observation. In this study, we are introducing a simple, but reliable stroke model using permanent middle cerebral artery occlusion in sheep. This model allows for control of ischemic lesion size and subsequent neurofunctional impact, and it is monitored by behavioral phenotyping, magnetic resonance imaging, and positron emission tomography. Neuropathologic and (immuno)-histologic investigations showed typical ischemic lesion patterns whereas commercially available antibodies against vascular, neuronal, astroglial, and microglial antigens were feasible for ovine brain specimens. Based on absent mortality in this study and uncomplicated species-appropriate housing, long-term studies can be realized with comparatively low expenditures. This model could be used as an alternative to existing large animal models, especially for longitudinal analyses of the safety and therapeutic impact of novel therapies in the field of translational stroke research.

Keywords

animal studies behavior MRI PET stroke stroke animal models

Introduction

Acute cerebral vessel occlusion causing ischemic stroke and leading to subsequent loss of cerebral tissue and function is one of the leading causes of death and disability in industrial nations (Kolominsky-Rabas et al, 2006). Systemic thrombolysis using tissue plasminogen activator or other agents can provide significant clinical benefits for stroke patients. However, only a minority of stroke victims can be treated with this approach, mainly because of the narrow time window for successful and safe intervention (Lapchak and Araujo, 2007). Consequently, the development of novel therapeutic strategies is essential to improve the situation in acute stroke treatment.

Current studies on experimental stroke therapies, evaluating the efficiency of neuroprotective agents, cell-based approaches, or other strategies, primarily use rodent models of permanent or transient focal cerebral ischemia. These animal models offer numerous advantages such as reproducible lesion size, detailed, but not capacious knowledge about pathophysiologic mechanisms of stroke, broad availability, and high flexibility for different experimental requirements. However, lesion size and reproducibility of sensorimotor deficits might differ between common rat strains (Traystman, 2003).

In the past, a noticeable number of experimental protocols based on neuroprotective drugs, which proved to be effective in rodents, have failed in clinical trials (Del Zoppo, 1995; Heiss, 2002). This indicates a clear demand for an additional experimental step between common rodent models and human patients in evaluating novel stroke therapies before entering clinical applications. Consequently, the STAIR criteria strongly recommend the use of appropriate, close-to-practice large animal models of focal cerebral ischemia (Schabitz and Fisher, 2006). In this regard, modern brain imaging techniques such as magnetic resonance imaging (MRI) and positron emission tomography (PET) are crucial for online monitoring of novel treatment protocols.

Human-relevant primate models, which are essential for investigating acute stroke, are restricted to specialized breeding facilities and, the application of marmoset models notwithstanding, do not often allow for long-term observation of experimental subjects because of high mortality (1999).

Therefore, the aim of this study was to develop a robust model of focal cerebral ischemia in sheep, which can easily be housed in a species-appropriate environment. Because of the rete mirabile, cerebral ischemia was induced by transcranial permanent middle cerebral artery occlusion (MCAO), providing reproducible neurologic dysfunctions and lesion sizes that can be modified by altering the number of middle cerebral artery (MCA) branches occluded. Moreover, the model provides options for detailed PET and MRI studies, using common imaging protocols. Additionally, we investigated the neuropathologic consequences of permanent MCAO in sheep, finding stroke-induced changes similar to those in human cerebral ischemia. No stroke-dependent mortality of experimental subjects was observed in the study.

Materials and methods

Experimental Animals

All experimental procedures were approved by the Experimental Animal Committee of the Regional Council of Leipzig. A total of 30 healthy outbreed adult hornless merino rams weighing 42 to 65 kg were obtained from the experimental farm of the Faculty of Veterinary Medicine of the University of Leipzig without any kind of selection. Subjects were kept in small flocks with ad libitum access to food and water except for 12 h before surgery and imaging procedures. All animals were allowed to enter a meadow for at least 5 h per day before surgery and from days 5 to 42 after MCAO. All medications and their application schemes used in the study are given in Supplementary Table 1.

Table 1

Behavioral assessment

Item	Score points
State of activity/consciousness
Normal	0
Apathy	1
Stupor (no further investigation possible)	(25)
Coma (no further investigation possible)	(27)
2. Food dehris remaining in mouth corner
Yes	1
No	0
3. Torticollis
Yes	1
No	0
4. Carpus and/or fetlock in partial flexion
Yes	1
No	0
5. Ataxia/dysmetria
None	0
Light ataxia (dysmetric limb movements)	1
Medium ataxia (animal staggers)	2
Severe ataxia (animal falls down)	3
6. Circlic movements
No	0
Occasionally	1
Permanent circling to left side	2
Permanent circling to right side wearing blindfold	+1
7. Right hemistanding reaction
No disturbance	0
Immediate adjustment using left forelimb, hindlimb without disturbance	1
Delayed adjustment using left forelimb, hindlimb without disturbance	2
Delayed adjustment using both left limbs	3
Delayed adjustment using left forelimb and right hindlimb	4
Delayed adjustment using right limbs	5
No adjustment right forelimb, delayed adjustment using right hindlimb	6
No adjustment in both limbs	7
8. Right hopping reaction
No disturbance	0
Delayed medial adjustment in forelimb	1
Delayed medial and lateral adjustment in forelimb	2
No medial, delayed lateral adjustment in forelimb	3
No medial or lateral adjustment in forelimb	4
No medial or lateral adjustment in forelimb, delayed or absent adjustment in hindlimb	5
9. Forced movement using only forelimbs (‘wheelbarrowing’)
No disturbance	0
Drifting to right side	1
Animal falls down	2
Maximum score points	27

Sheep were examined regularly for neurological dysfunctions according to the items given. The score point system is based on a common veterinary neurological investigation technique (Oliver et al, 1997). In the case of stupor and coma, animals received 25 and 27 score points, respectively. No further investigation could be performed. It should be noted, that stupor was detected once on day 1, while a comatose state was not caused by MCAO in any animal observed in this study.

Induction of Stroke by Middle Cerebral Artery Occlusion

Rams were randomly assigned to one of five groups: transcranial 1-branch- (n = 5), 2-branch- (n = 5), or total proximal MCAO (n = 10), sham operation (MCA touched but not occluded, n = 5), and control group (no operation, n = 5).

Anesthesia was performed according to Supplementary Table 1. Animals were intubated after induction of anesthesia. An intraesophageal tube was used to avoid ruminal tympania. Throughout the surgical procedure, tracheal respiration with 1.5% to 2.0% isoflurane in pure oxygen was performed. An electrocardiogram was recorded, and blood pressure was continuously measured using an intra-arterial probe placed in the dorsal pedis artery of the left hind limb.

The relevant anatomic structures and the surgical procedures are shown in Figure 1. Briefly, the skin between the ear and eye was incised on the left side of the head to expose the branches of the superficial temporal artery and the accompanying vein. After occlusion of these vessels by high frequency bipolar forceps (Liga Sure, Valleylab, Boulder, CO, USA), the origin of the temporalis muscle was incised at the temporalis line and the muscle was carefully elevated from the parietal bone. After exposure of the skull bone surface, a hole was drilled into the parietal plate using a set of neurosurgical burrs and was then extended using Kerrison rongeurs. On local incision of the dura mater, MCA or its branches (according to the experimental group) were permanently occluded. Then, the temporalis muscle was fixed on the temporalis line and the skin was sutured using 2-0 absorbable filaments (Ethicon Ltd, Norderstedt, Germany). After termination of surgery, animals were disconnected from artificial ventilation and immediately taken to a specialized awakening box. Intratracheal and intraesophageal tubes were removed when the swallowing reflex was restored. A postsurgical antibiotic treatment (Supplementary Table 1) was performed for 7 days.

Figure 1

Intraoperational situs, topographical anatomy and cerebral blood supply. Principle of the surgical procedure and the most important anatomic structures in the operational field. (A) gives 3D CT reconstruction of a sheep skull (obtained in planning experiments before the study). (B) to (E) show illustrations drawn from series of photographs focusing on important structures. (1) mCa supply area (schematic drawing of branches and capillaries); (2) rete mirabile epidurale rostrale; (3) approximate location of total MCAO; (4) ramus caudalis ad rete mirabile epidurale; (5) maxillary artery (note: all structures are projected on lateral bone surfaces but can be found behind these surfaces); (6) temporal muscle; (7) A. auricularis caudalis; (8) N. facialis (N. VII); (9) N. auriculopalpebralis; (10) A. temporalis superficialis; (11) A. carotis externa; (12) drill hole; (13) Sulcus centralis; (14) MCA. Blood supply of the sheep brain is illustrated in (F) to (H). Pictures show a basal view of the circulus arteriosus and are adapted from Förschler et al, 2007. (F) A corrosion cast made by intra-arterial delivery of Mallocryl M (Laborchemie Apolda, Jena, Germany) at day 43 after MCAO. Because of the MCAO location (white arrow head) the distal supply area of the left MCA is not filled with Mallocryl M. (G) Time of flight MRA of a control animal not subjected to MCAO. (H) The corresponding scheme drawing. (15) A. cerebri anterior (rostralis); (14) MCA; (5) A. maxillaris, which supplies the (2) rete mirabile epidurale rostrale; (16) A. basilaris; (17) A. choroidea rostralis.

Imaging Procedures

Magnetic resonance and PET examinations were performed on days 1, 14, and 42 after onset of stroke. Magnetic resonance imaging was performed using a 1.5 T clinical whole body scanner (Gyroscan Intera; Philips, Koninklijke, The Netherlands) with a flexible double loop RF-coil (Sense Flex M; Philips). Animals were anesthetized as described in Supplementary Table 1. Spontaneous ventilation of sheep was monitored by a pneumatic respiration probe connected to the scanner. Magnetic resonance imaging was performed according to protocols shown in Supplementary Table 2. Altogether, T1 turbo spin echo, T2 turbo spin echo, T2 diffusion weighted (DWI), T2 FLAIR, T2* gradient echo, and time of flight MR angiography (MRA) sequences were recorded. To diminish the susceptibility artifacts in the DWI and to shorten the examination duration, a parallel imaging protocol was used (SENSitivity Encoding; Philips) with a SENSE factor of 1.5 or 1.3 (MRA only). Three diffusion directions and two b-values, 0 and 1000, allowed for calculation of apparent diffusion coefficient maps by means of the standard software implemented on the scanner.

For MRI volumetry, T2 weighted sequences were analyzed by ImageJ 1.34 secs software (Wayne Rasband; National Institutes of Health, Bethesda, MD, USA), whereas the YAWI 2D 1.3.2. software (open source) served as the selection tool. The sizes of the ischemic lesion (area of cytotoxic edema as verified by decreased apparent diffusion coefficient values of the DWI), of the ipsilateral and contralateral side ventricles, and of both hemispheres were all calculated. Infarct volume was assessed by calculating the size of the lesion observed in T2W MR images on day 1. On days 14 and 42, the difference between the volumes of both side ventricles was added to the size of the lesion (if observed) to compensate for occasionally occurring difficulties in discrimination between the lesion area and side ventricle in T2 weighted images.

Tissue loss because of ischemic damage was represented by the hemispherical atrophy. For this process, the quotient between the size of the ipsilateral and contralateral hemispheres minus the associated ventricle volumes was calculated on days 14 and 42.

Positron Emission Tomography Imaging

In addition to MRI, serial PET imaging was performed in a subgroup of five sheep with total proximal MCAO. This was performed using a high-resolution clinical scanner (ECAT EXACT HR+; Siemens/CTI, Knoxville, USA). The scanner characteristics include: axial field of view 15.5 cm, 63 parallel transverse slices, slice thickness 2.4 mm, final reconstructed image resolution 7.1 mm (transverse), and 6.7 mm (axial). Cerebral blood flow (CBF) was visualized and quantified in the sheep using ¹⁵O-H₂O. Further, on day 42 cerebral metabolic rate of glucose (CMR_Glu), a surrogate measure of brain viability was obtained by means of ¹⁸F-FDG PET. In addition to these five sheep with total MCAO, a series of one subject each with 1-branch and 2-branch MCAO and sham surgery underwent brain CBF PET with ¹⁵O-H₂O 1 day after surgery. The anesthetized sheep were i.v. administered with ∼1000MBq ¹⁵O-H₂O and ∼370MBq ¹⁸F-FDG, respectively. In the case of ¹⁵O-H₂O, dynamic PET data were acquired in 3D-mode over 5 mins, and in the case of ¹⁸F-FDG in 2D-mode over 60 mins. Further details on the PET acquisition parameters are given in Supplementary Table 3. After standard image data reconstruction and correction for measured tissue attenuation, the resulting PET image datasets were transferred to a Hermes workstation (Hermes Medical Solutions, Stockholm, Sweden). By using the multimodality software, irregular regions of interest (ROI) were manually defined by an experienced investigator in all MCAO-affected transverse slices of the five sheep brains after total MCAO for the areas with uptake deficits. Slice-wise, these ROI were horizontally mirrored to the contralateral (nonaffected) brain hemisphere to define reference ROI. From these ROI, the volume as well as the relative (as related to the mirrored contralateral VOI) severity of the MCAO-induced activity deficits were calculated.

Behavioral Phenotyping

All animals were examined for neurofunctional disabilities before surgery (sham and MCAO) as well as on days 1, 4, 7, 10, 16, 25, 32, and 42 thereafter. For this process, a neurologic score point system was developed. The system is based on the common test system for neurologic dysfunctions in large animals (Oliver et al, 1997), however, focuses on the specific functional deficits observed in most animals after MCAO. A detailed description of the parameters investigated is given in Table 1. Interestingly, sheep rapidly habituated to weak or medium nociceptive stimulation. Therefore, sensor function could not be tested reliably.

Neuropathology, Immunohistochemistry and Immunofluorescence

Animals were killed 43 days upon MCAO by a single i.v. injection of 15 mL pentobarbital-Na (Eutha 77; Essex Pharma Ltd, Munich, Germany). After immediate decapitation heads were perfused through internal carotid arteries with 1 L of phosphate buffered saline solution (pH 7.4) followed by 14 L of 4% paraformaldehyde (Sigma Aldrich Ltd, Taufkirchen, Germany)/0.1 mol/L phosphate buffered saline. Occipital and parietal skull plates were removed followed by immersion fixation in 4% paraformaldehyde/0.1 mol/L phosphate buffered saline overnight before the brain was removed.

After gross examination, the whole brain was coronally cut into equally spaced slices (approximately 4 mm) for macroscopic and microscopic examination. Two specimens were taken from three defined sites of the left hemisphere (Figure 5A): the macroscopically assessed frontal and occipital borders of the infarct and the level of the left MCA. From the intact right hemisphere, two samples corresponding to locations 1a and 1b were taken. The coronal sections were fixed in buffered 4% paraformaldehyde for another 2 days, processed routinely, embedded in paraffin, and cut in 4 μm thick sections. For conventional light microscopy sections were stained with hemalaun and eosin or pikrosirius red by standard methods and facing cut surfaces the consecutive specimens were histologically examined.

For immunohistochemistry, the peroxidase antiperoxidase method was used. Sections were dewaxed, rehydrated, and treated with hydrogen peroxide 0.5% in methanol for 30 mins at room temperature (RT). After washing in Tris-buffered saline (TBS), blocking with 50% pig serum in TBS at RT was performed for 10 mins. The polyclonal antibody rabbit anti-bovine glial fibrillary acid protein (GFAP; 1:500) was applied to the sections overnight at 4°C and the slides were incubated for 30 mins at RT with pig anti-rabbit immunoglobulin G secondary antibody (DAKO Cytomania GmbH, Hamburg, Germany; 1:100) thereafter. This was followed by incubation for 30 mins at RT with the rabbit-peroxidase antiperoxidase-complex (DAKO; 1:100). After signal detection with a freshly prepared solution of 3,3‘-diaminobenzidintetrahydro-chloride (Fluka Feinchemikalien, Neu Ulm, Germany) for 10 mins at RT, the sections were counterstained with Papanicolaou's stain. For control purposes the primary antibody was replaced by normal rabbit serum (DAKO). Histologic and immunohistochemical investigations were performed using a standard microscope (Olympus).

For fluorescence microscopy perfusion fixated coronal sections taken from the reactive zone next to the lesion and a corresponding area in the intact hemisphere were cryopreserved in 30% sucrose (Sigma Aldrich)/0.1 mol/L phosphate buffered saline. Free floating sections from cryopreserved specimens were extensively rinsed with 0.1 mol/L TBS, pH 7.4. Nonspecific binding sites for subsequently applied antibodies and lectins were then blocked with 5% normal donkey serum in TBS containing 0.3% Triton X-100. Next, the sections were incubated with primary antibodies and biotinylated lectins (see Supplementary Table 4) overnight at RT. For triple fluorescence labeling, markers were simultaneously applied in two main series: (1) mouse-anti-CD11b + rabbit-anti-GFAP + biotinylated RCA or (2) biotinylated tomato (or potato) lectin + rabbit-anti-GFAP + mouse-anti-NeuN. After rinsing with TBS, the sections were processed by concomitant use of appropriate carbocyanine (Cy)2-, Cy3-, and Cy5-conjugated secondary immunoreagents (all Dianova, Hamburg, Germany; supplier for Jackson Immuno Research, West Grove, PA, USA) and used at 20 mg/mL for 1 h. Finally, sections were extensively washed with TBS, briefly rinsed with distilled water, mounted onto fluorescence-free-slides, air-dried and coverslipped with Entellan (Merck, Darmstadt, Germany).

In control experiments, the omission of primary antibodies or lectins resulted in the expected absence of any cellular labeling, switch of fluorophores caused no alteration of staining patterns.

All fluorescence slides were inspected with a confocal laser-scanning microscope LSM 510 Meta (Zeiss, Jena, Germany) equipped with an argon laser exciting Cy2 at 488 nm and two helium-neon lasers (543 nm for Cy3 and 633 nm for Cy5). Cy5-labeled structures were color-coded in blue.

Statistics

Statistical analysis was performed using the SigmaStat 3.0 software (Systat Inc., San Jose, CA, USA). Differences in neurofunctional outcome between different MCAO groups and controls were evaluated using the one-way ANOVA test. Differences in MRI and PET findings size were analyzed by the Student's t-test in the case of normally distributed data.

A P-value < 0.05 was considered statistically significant and P-values < 0.01 as highly statistically significant. The levels of significance are indicated by single (P < 0.05), double (P < 0.01), or triple (P < 0.001) asterisks. If not stated otherwise, the data are presented as mean±s.d.

Results

General Observations and Animal Welfare

No animal died because of reasons related to the surgical procedure or stroke. There was a reversible reduction of vigilance and food intake for a time period of up to 4 days after MCAO. Although movement disturbances including hemiparesis were observed, especially in the total MCAO group, hemiplegia was not caused in any animal. Hence, the rams were able to stand, move about, and join other sheep. Outdoor housing was possible from day 5 onwards.

Diffusion Disturbances and Hemispherical Atrophy in Relation to Numbers of Middle Cerebral Artery Branches Occluded

Middle cerebral artery occlusion but not sham surgery caused diffusion disturbances and atrophy in the sheep brain.

The development of diffusion disturbances and hemispherical atrophy is illustrated in Figures 2 and 3. In the control or sham-treated animals, MRI revealed no signs of ischemic damage. However, in three of the five sham animals diffusion disturbance (Figure 2A, white arrow heads) were observed on day 1, but not thereafter. There was no indication for an ischemic origin of these disturbances in the corresponding apparent diffusion coefficient maps. Furthermore, these areas appeared as small cortical contusion damages in histologic examinations.

Figure 2

Lesion size and hemispherical atrophy in relation to MCAO modality. (A) Different lesion sizes were observed in MR and PET imaging in this study on day 1. Lesion size was largest after total MCAO. To exclude spontaneous reopening of occluded branches, MRA was performed on day 14. In sham-operated animals, which showed mild and transient neurofunctional disabilities in single cases (compare Figure 3), no major signs of ischemia were observed. However, in cortical areas next to the drill hole, indications of slide perfusion dysbalances appeared corresponding to vasogenic edema in DWI maps (white arrow heads) as well as in perfusion PETon day 1, but not thereafter. Hence, these findings were interpreted as small contusion damages without permanent loss of tissue or motor function. Corresponding MRA showed no signs of vessel occlusion. (B) Diffusion disturbance in T2W MR images. In correlation to the findings on day 1, the size of the diffusion disturbance was largest after total MCAO and differed between the groups on all days of investigation (P < 0.05 or less). (C) Furthermore, hemispherical atrophy was calculated on days 14 and 42. Tissue loss was most prominent upon total MCAO on day 42 and larger after 2-branch McAo compared with 1-branch MCAO (P < 0.05). No differences were observed on day 14. No ischemic lesion or tissue loss was detected in sham-operated animals.

Figure 3

Follow-up ¹⁵O-H₂O PET imaging. (A) Transverse brain perfusion PET slices as correlated with T2-MRI (TSE on days 1 and 14, FLAIR on day 42) slices on days 1, 14, and 42 in a representative sheep after total proximal MCAO. Group statistics of CBF deficit volumetry (B) and calculation of relative CBF in MCAO-related deficits (C) in follow-up ¹⁵O-H₂O-PET of five sheep after total proximal MCAO. For description of the ROI technique used, see Materials and methods.

Magnetic resonance imaging analyses revealed that the volume of diffusion disturbances reflected the number of MCA branches occluded. Thereby, the initial (day 1) lesion volume in diffusion-weighted MRI after total MCAO was significantly larger compared with the volume after 2-branch-MCAO (16.3±5.2 versus 8.7±3.9 mL, P < 0.01; Figure 2B). The 1-branch-MCAO led to significantly less lesion volumes (5.6±3.6 mL) as compared with the 2-branch MCAO (P < 0.05; Figure 2B). Over time, the volume of the diffusion disturbance decreased for all experimental subgroups in a parallel manner, that is, the significant differences in lesion volumes between the experimental subgroups was maintained on days 14 and 42 (Figure 2B).

Concerning the ratios between the volumes of both hemispheres (lesion versus intact; Figure 2C), there was no statistically significant difference between the experimental subgroups on day 14 (P > 0.38 or higher). However, on day 42 these atrophy ratios were significantly higher for the total MCAO subgroup in comparison to the 2-branch-MCAO group (P < 0.05), whereas 1-branch-MCAO resulted in significantly less atrophy ratios compared with 2-branch-MCAO (P < 0.05; Figure 2C).

15O-H2O and 18F-FDG Positron Emission Tomography Show Characteristic Middle Cerebral Artery Occlusion-Related Deficits

Figure 3 shows the influence of the extent of MCA surgery on the stroke-related CBF deficits as visualized by ¹⁵O-H₂O PET. In accordance with the MRI data, the deficit extent increased in the order sham surgery < 1-branch MCAO < 2-brach MCAO < total MCAO (Figure 2A). For the five sheep with total MCAO which underwent serial brain perfusion PET imaging, the extent of the CBF deficits defined by the described ROI technique increased over time from 14.3±1.3 mL on day 1 (over 9.5±6.3 mL on day 14; not significant) to 19.5±3.7 mL on day 42 (P < 0.05; Figure 3B). The relative (compared with contralateral hemisphere) severity of the CBF deficits caused by the MCAO was 42.6%±1.7% on day 1, 27.8%±5.6% on day 14, and 37.2%±6.4% on day 42 (P < 0.05 for day 1 versus day 14 and day 1 versus day 42; Figure 3C). Furthermore, in these sheep the final CMR_Glu deficit on day 42 had an extent and relative severity of 10.7±1.8mL and 31.4%±3.2%, respectively (data not shown).

Behavioral Phenotyping Shows Functional Disabilities Correlated to Lesion Size

Assessment of motor functions and vigilance resulted in reproducible defect patterns corresponding to the size of the ischemic lesion induced by MCAO (Figure 4). Total MCAO was accompanied by more severe dysfunctions compared with other groups throughout the observation period (P < 0.05 or less). The 1-branch- and 2-branch MCAO groups could be discriminated by score differences until day 10 (P < 0.01 or less) but not thereafter. In all experimental groups there was a tendency toward partial regain of motor functions. Therefore, 1-branch- and 2-branch-MCAO, but not total MCAO, caused only minor permanent deficits such as mild torticollis and slightly delayed startle reflexes. Permanent deficits caused by total MCAO included torticollis, occasional circle movement, and clearly delayed hemistanding and hopping reactions.

Figure 4

Clinical assessment using neurologic score point system. Behavioral phenotyping after MCAO showed statistically significant differences between the experimental groups in relation to the number of MCA branches occluded. There was a certain recovery in all occlusion groups, without reaching baseline levels. Sham-operated animals showed only mild and transient dysfunctions on days 1 and 4, whereas 1- and 2-branch-MCAO yielded significantly different clinical scores until day 10 (P < 0.01) and resulted in only minor dysfunctions at the end of the study. Total MCAO caused the most severe disabilities throughout the observation period (P < 0.05).

In sham-operated animals, minimal transient dysfunctions were observed only on days 1 and 4. Those dysfunctions were rather unspecific, mainly including a positive item 2 (Table 1) and a reduced state of activity. Therefore, they could not be discriminated from common postsurgical effects, not related to MCAO. The transient dysfunctions in sham-operated animals were significantly less (P < 0.001) compared with all groups subjected to MCAO. At no time point any dysfunctions were observed in nonoperated control animals.

Gross pathology, Immunohistochemistry, and Fluorescence Microscopy

The infarct area in the left hemisphere was clearly discernible as a concave light brown area (Figure 5B) of softer consistency with a slightly thickened and opaque leptomeninx. The cerebral gyri and sulci were hardly visible or already missing. In the coronal sections tissue shrinkage accompanied by a varying enlargement of the associated lateral ventricle (Figure 5C) was obvious. The sharply demarcated infarct area, consistently involving cortex and medullary layer, showed variable cavitations. Apart from the extension, histopathologic findings did not differ between the MCAO groups and can be described as follows.

Figure 5

Histopathologic sampling procedure and gross pathology. All brain specimens investigated were obtained after killing the animals on day 43 after MCAO. (A) Schematic drawing of the sampling procedure for histologic purposes. The infarction is delineated by a white line. 1a and 1b: Level of the (occluded) MCA; 2a and 2b: Frontal border of the infarct; 3a and 3b: Occipital border of the infarct. (B) Gross findings. Infarction (delineated by an orange line) appears as concave, light brown, well delineated area, and thickened, opaque leptomeninx (day 42, brain perfusion- and immersion-fixed). (C) Caudal surface of a coronal section taken from the level of the occluded MCA: a well-delineated, yellow-brown infarction area in the left hemisphere and enlargement of the ipsilateral side ventricle (day 42, perfusion- and immersion-fixed tissue). (D) Schematic drawing of the tissue alterations given in C. Infarct area (1), enlarged left lateral ventricle (2) and normal-sized right lateral ventricle (3). (E) Corresponding T2 FLAIR image from day 42.

In the area of MCAO, the leptomeninx displayed a mild to focally moderate active fibrosis with scattered mononuclear infiltrations. In the infarct border, fibrotic changes were moderate and proliferation of capillaries was visible. The classification of the infarct area into a central zone (necrosis), a reactive zone (directly bordering the necrosis), a marginal zone (transitional zone with both intact and affected brain tissue), and a remote zone was adapted from the classification of Garcia and Kamijyo (1974).

The central zone (Figures 6A and 6B) consisted of abundant foamy fat granule cells, scattered lymphocytes and plasma cells, few siderocytes, minimal to moderate angiogenesis, and connective tissue proliferation, increasing next to the leptomeninx. Cavitation was noted in the depth of infarct (Figure 6A). The formation of connective tissue and angiogenesis increased to the margins of the central zone.

Figure 6

Histopathologic findings. Histopathologic evaluation was performed from brain specimens obtained on day 43 on induction of ischemia. (A) The area of former liquefactive necrosis (infarction) with the central zone (ce) containing numerous fat granule cells (as given in the inset). Angiogenesis and cyst formation (asterisk) was observed. In the reactive zone (re), moderate gliosis, angiogenesis, and connective tissue proliferation (black arrow head) was found, whereas in the marginal zone (ma) gliosis became evident (H & E-stain, scale bar = 200 μm, inset: scale bar = 20 μm). (B) Extensive astroglial reactivity was observed after GFAP-immunostaining (Nomarski-interference contrast, scale bar = 200 μm, inset: scale bar = 20 μm). In corresponding areas of the contralateral, intact hemisphere (C) only very few neuronal hypoxic cell changes (inset: black arrow heads) and very mild gliosis were found (H & E-stain, scale bar= 200 μm, inset: scale bar = 50 μm). (D) This was also verified by GFAP staining in the contralateral hemisphere (Nomarski-interference contrast, scale bar = 200 μm, inset: scale bar = 20 μm).

In the reactive zone (Figures 6A and 6B; see also Figure 7) moderate augmentation of astrocytes and microglia as well as moderately increased capillarization was observed. In close proximity to the central zone neurons were completely missing. More distant, neurons showed mild satellitosis and signs of hypoxic cell changes. In the medullary layer, spheroids occurred in mild to moderate, focally high numbers.

Figure 7

Confocal fluorescence microscopy. Fluorescence immunohistochemistry was performed using brain tissue that was cryopreserved immediately after animal euthanasia on day 43. Augmentation of cD11b⁺ microglial cells (lb>A) next to LEA⁺ vessels (B) was observed in the reactive zone (merging: C). Blood vessel staining was performed by tomato lectin (B). Triple staining as shown in (D) to (G) likewise showed augmentation of astrocyte reactivity as indicated by green GFAP fluorescence (E) in these areas. Micrographs of the reactive zone close to the central zone are given in (H) to (K) whereas (L) to (O) show pictures from a corresponding area in the intact hemisphere. Note the massive loss of neurons indicated by diminution of NeuN⁺ cells and the consecutive augmentation of GFAP⁺ astrocytes forming a glial scar adjacent to the central zone. In the intact hemisphere, astrocytes show only minor morphologic signs of reactivity, whereas the density of NeuN⁺ neurons is not changed in this area. Scale bars (A to K) = 100 μm, (L to O) = 75 μm.

The cortex of the marginal zone (Figures 6A and 6B) showed, hinted to minimal satellitosis, mild hypoxic neuronal changes, astrocytosis, as well as moderate microgliosis. In the medullary layer, showing mild astrogliosis and microgliosis, spheroids were rare.

In the remote zone (Figures 6A and 6B), the cortex displayed mild satellitosis, hypoxic neuronal cell changes and slightly increased numbers of astrocytes and microglia.

The intact right hemisphere showed mild leptomeningeal fibrosis with few mononuclear infiltrations. The cortex and medullary layer displayed alterations already described for the remote zone of the affected left hemisphere.

All antibodies and lectins used in the described protocols labeled distinctive cellular structures in sheep brain specimens. Investigation of the reactive zone using fluorescence microscopy showed a moderate increase of CDl1b⁺ cells, colocalized with vessel structures (LEA; Figures 7A–C). Triple staining experiments including antibodies directed against GFAP indicated enhanced astrocytosis in areas where augmentation of CDl1b⁺ cells was detected (Figures 7D–G).

In border zone areas next to the core of the lesion, reactive astrocytosis was indicated by strong GFAP-staining (Figure 7H) representing glial scar tissue. Although the density capillaries was slightly increased as revealed by RCA (Figure 7I) or using Solanum tuberosum agglutinin (not shown) there was a consecutive loss of NeuN⁺ neurons in areas next to the central lesion (Figure 7K). NeuN⁺ cells were detected in normal density only in areas of reduced GFAP-immunolabeling (Figure 7L).

In corresponding areas of the intact hemisphere and regions more distant form the central lesion (Figures 7M–P), there were no local differences between the density of NeuN⁺ cells (Figures 7O and 7P) whereas slight astrocytosis indicated by GFAP ⁺ (Figure 7M) cells could be observed. However, astrocytosis was much less prominent in corresponding areas of the intact hemisphere than next to the lesion.

Discussion

The treatment protocols for ischemic stroke, which are currently available for routine clinical application and are based on thrombolysis, are efficient but limited by a narrow time window. It is well known that several alternative approaches, including neuroprotective and cell based strategies, have been shown to improve stroke outcome and possibly to widen the therapeutic time-window in rodents (Newcomb et al, 2006; Daadi et al, 2008); however, these approaches have still not been successfully translated into routine clinical protocols and additional research, especially elucidating underlying mechanisms, will be urgently needed to assess the value of these experimental approaches for human stroke patients. It is further known from previous trials involving neuroprotective substances that appropriate animal models are crucial for verifying novel, promising approaches. An optimal preclinical stroke model should reflect the size and complexity of the human brain and in parallel allow for the monitoring of clinical, pathophysiologic, morphologic, and histologic parameters of stroke development (Green, 2002).

In principle, most primate models fulfill these requirements and are of outstanding importance in acute stroke research but are often accompanied by a limited observation time because of high mortality rates. However, studies lasting for 10 weeks using total MCAO in marmosets (Marshall et al, 2003), one of the smallest primate species, were not able to predict clinical ineffectiveness of the neuroprotective agent NXY-059 (Shuaib et al, 2007). This might possibly be related to the small size of the marmoset brain. Thus, neuroprotective agents (as in rodents) could potentially reach large relative proportions of hypoxic or ischemic tissue by diffusion and/or active molecular transport processes within the therapeutic time frame after permanent MCAO, resulting in an average volume of protected tissue of 90 mm³, also accompanied by reduced functional deficits (Marshall et al, 2003). This absolute amount of rescued brain tissue would probably not be of that particular functional relevance in larger species or humans also experiencing extended stroke because of total MCA blockage.

Limited observation times after MCAO which are seen in many currently available large animal models could be resolved by using ruminants such as sheep, which also exhibit important similarities to primates in terms hematologic parameters and blood grouping (see Supplementary Table 5). The absent of stroke-related mortality in our study was most probably because of the partial removal of parietal skull bone thus limiting the increase of intracranial pressure in the phase of the subacute cytotoxic brain edema. Also, the induction of cortical (distal) but not total (proximal) MCAO as performed in this study, effectively limits the increase of intracranial pressure.

However, it has to be stated that more methodological tools for ovine studies, especially those aiming on metabolic and molecular processes after cerebral ischemia, have to be developed. This is crucial to augment the value of the model for stroke research and to bring the level of knowledge on these processes in sheep to a level that is common in other, well-established large animal models, in particular primates. Although sheep models of global cerebral ischemia are broadly used for pathophysiologic (Riddle et al, 2006) and methodical (Martinez-Coll et al, 2003) investigations, to our knowledge, this is the first description of an ovine model of focal cerebral ischemia.

Different investigation modalities were used to evaluate the consequences of MCAO in sheep, including behavioral testing, MR and PET imaging, and histopathologic methods.

In behavioral tests, long-lasting, severe motor functional consequences were only observed after total MCAO. Thus, total MCAO might be required to detect beneficial therapeutic effects on clinical outcome in sheep at later time points. However, even after total vessel occlusion, none of the 10 animals died whereas all subjects were able to join their flock on a meadow within 5 days upon MCAO. However, clear signs of cerebral infarction in brain imaging as well as histologic and cellular consequences could also be induced without permanent major clinical consequences by 1- or 2-branch occlusions. These occlusion modalities might be used to study the pathophysiology of stroke without the necessity of focusing on clinical outcome parameters. A possible limitation of the sheep model is the fact that the animals rapidly habituate to nonendangering nociceptive stimuli. This aggravates assessment of MCAO-induced sensory deficits. However, these deficits can be at least partially evaluated indirectly by observing ataxic movements and disturbed startle reflexes.

In clinical routine, MRI is currently the imaging method of choice in diagnosis (Chalela et al, 2007) and follow-up of ischemic stroke in humans and animals (Back et al, 2004). Size and weight of sheep provide an easy access to clinical MR scanners. Using not only morphologic, but also functional MRI tools, it is possible to verify successful induction of stroke by DWI (van Everdingen et al, 1998) at an early time point. Brain MR imaging of sheep indicated diffusion disturbances, which in their extent, localization, and time-dependency during a 42-day-timecourse are comparable to those observed in human patients (Forschler et al, 2007, who for comparison referred to Ritzl et al, 2004). Magnetic resonance imaging is, furthermore, able to depict infarct size and associated vessel occlusion type in vivo (Higashida et al, 2003). Due to exposing of the MCA trifurcation by the surgical approach introduced in this study, it is possible to create infarcts of different volumes and high reproducibility. Although the number of branches occluded could reliably be verified via MRA (Figure 2A), lesion size measured by MRI reflected the numbers of MCA branches occluded on day 1 (Figure 2B). Subsequent hemispherical atrophy also correlated with the number of cauterized MCA branches (Figure 2C). In addition, MRI not only allows for animals to be compared at several time points, but also permits lesion development and possible therapy effect to be followed in vivo within the same subject by repeated investigation, which yields reliable statistics even in heterogeneous study groups. For these reasons, the uncomplicated use of MRI enables the model to stay as near as possible to the clinical settings used for human patients.

As with the extent of the brain diffusion disturbances (as seen by MRI), it was possible to control the extent and severity of the CBF deficits by varying the number of occluded MCA branches. This was clarified by means of ¹⁵O-H₂O PET imaging on day 1 after MCAO (Figure 2A). Follow-up brain perfusion PET on day 14 and 42 in a subset of five sheep after complete MCAO revealed a final (after 42 days) increase of the CBF deficit volume, whereas the relative severity of the CBF deficit in the stroke-related lesion returned to the baseline level on day 1. In the subacute stage after stroke (14 d), the CBF deficits showed a marked improvement of both defect extent and severity. The additional PET imaging of brain glucose consumption with ¹⁸F-FDG, a surrogate marker of brain viability, after 42 d revealed CMR_Glu deficits which were 55% of the size and 84% of the severity of the corresponding CBF deficits. Our PET results are a good approximation of the known CBF time course and CMR_Glu outcome after ischemic stroke in human patients (Huber et al, 1992). In particular, our finding of a temporal CBF improvement in the MCAO-related brain tissue might fit to the established concept of reversible postischemic hyperperfusion (Marchal et al, 1999).

Overall, a possible limitation of the sheep model at this point is that extensive anesthesia, performed for 8 h or longer is accompanied with reduced recovery rates in sheep thus possibly limiting the investigation of the development of the ischemic lesion in sheep beyond this time window after MCAO in case recovery is needed for subsequent investigations.

Recording of electroencephalograms or somato-sensory-evoked potentials was not performed in this study but was previously shown to be feasible in nonanesthetized sheep (Voss et al, 2006, Marcus et al, 1997), thus allowing electrophysiologic studies using the species to be performed as well.

Macroscopic post mortem evaluation of brains resulted in lesions that appear to be similar to those observed in human stroke victims and varied in size according to the number of occluded MCA branches. Microscopic neuropathologic evaluation showed typical signs of ischemia that can be found among all species including primates (Gao et al, 2006). Among these signs, neuronal loss and degeneration, cavitation in the central lesion areas, macrophage recruitment, and angiogenesis were most evident. Neuropathologic findings showed that the outcome in the sheep model is very close to the human situation, giving an appropriate surrogate to human neuropathology on cerebral ischemia.

As sheep are not frequently used for medical research at the present time, there is a lack of species-specific antibodies. However, all antibodies used in this study (Supplementary Table 4) showed sufficient cross-reactivity and could, therefore, be used to evaluate cellular alteration in the adult sheep brain after ischemic stroke induced by MCAO. Although the usefulness of other antibodies, for example directed against oligodendroglia, still has to be elucidated, the immunolabeling shown is feasible for investigating at least basic processes after ischemic stroke in sheep with or without experimental therapeutic interventions. Double and triple fluorescence staining revealed typical cellular reaction patterns expected in the mammalian brain on ischemic stroke such as neuronal loss close to the lesion, astrogliosis, and microglial augmentation.

A disadvantage of the model is the longer generation time of sheep compared with laboratory rodents and the dependency on the reproductive season. The merino rams used in this study needed to mature for approximately 12 to 18 months before they were considered to be adult animals. This limits the flexibility of the sheep model. However, the animal species used in existing feline and canine models show similar growth times, whereas adulthood in most primates is reached even later. Another disadvantage is the necessity of a reliable, but rather complex transcranial surgical approach for MCAO because of the rete mirabile (Figures 1F–H), preventing success of intraluminal methods of vessel occlusion.

It has been shown that the ovine stroke model provides major advantages, making it an attractive tool especially for long-term evaluation of novel therapeutic concepts. As such, this new large animal model could be appropriate for verifying new stroke therapies in translational medicine and could, for example, be of particular value for evaluation of the safety and efficacy of autologous cell therapies, which can hardly be evaluated in small species because of the limited number of obtainable cells per animal.

Overall, the sheep model of stroke, by overcoming some major limitations of other large animal models, has the potential to improve our understanding of stroke pathophysiology and to support the development of novel stroke therapies.

Disclosure/conflict of interest

No conflict of interest is evident with respect to this study.

Footnotes

Acknowledgements

The authors thank the MRI, PET and cyclotron crews at the Departments of Neuroradiology (Professor Kahn, Dr Lobsien) and Nuclear Medicine (Professor Sabri, Dr Grofimann) at the University of Leipzig. Furthermore, the authors especially thank the Departments of Veterinary Anatomy and Large Animal Surgery for support; Dr Grosche for confocal fluorescence micrographs and Mrs Ute Bauer for technical assistance. The authors are grateful to Mr Scott Krausen, a zoologist and artist who drew the illustrations in .

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