Spontaneous ischaemic stroke lesions in a dog brain: neuropathological characterisation and comparison to human ischaemic stroke
- Barbara Blicher Thomsen1,
- Hanne Gredal1,
- Martin Wirenfeldt2,
- Bjarne Winther Kristensen2,
- Bettina Hjelm Clausen3,
- Anders Elm Larsen3,
- Bente Finsen3,
- Mette Berendt†1Email author and
- Kate Lykke Lambertsen†3, 4, 5
© The Author(s) 2017
Received: 19 July 2016
Accepted: 31 December 2016
Published: 13 January 2017
Dogs develop spontaneous ischaemic stroke with a clinical picture closely resembling human ischaemic stroke patients. Animal stroke models have been developed, but it has proved difficult to translate results obtained from such models into successful therapeutic strategies in human stroke patients. In order to face this apparent translational gap within stroke research, dogs with ischaemic stroke constitute an opportunity to study the neuropathology of ischaemic stroke in an animal species.
A 7 years and 8 months old female neutered Rottweiler dog suffered a middle cerebral artery infarct and was euthanized 3 days after onset of neurological signs. The brain was subjected to histopathology and immunohistochemistry. Neuropathological changes were characterised by a pan-necrotic infarct surrounded by peri-infarct injured neurons and reactive microglia/macrophages and astrocytes.
The neuropathological changes reported in the present study were similar to findings in human patients with ischaemic stroke. The dog with spontaneous ischaemic stroke is of interest as a complementary spontaneous animal model for further neuropathological studies.
KeywordsAnimal model Astrocyte Canine Cerebral infarction Cerebrovascular accident Infarct Microglia Middle cerebral artery occlusion
Dogs suffer from spontaneous ischaemic stroke with neurological signs and magnetic resonance imaging (MRI) findings largely comparable to those of humans [1, 2]. Like humans, dogs with ischaemic stroke display variable neurological signs depending on the topography of the vascular occlusion and the size of the infarct [1, 3–5].
Experimental rodent models have provided extensive knowledge of the pathophysiological mechanisms of ischaemic stroke [6, 7]. It has, however, proved difficult to translate results obtained from such models into successful therapeutic strategies in human stroke patients [8, 9]. In order to face this apparent translational gap within stroke research, it has been proposed to search for alternative animal models comprising more aspects of the human disease .
Dogs resemble humans with regard to basic anatomy of a large-sized gyrencephalic brain, its vascularization and a high ratio of white compared to grey matter [11–13]. Furthermore, dogs age naturally and, as humans, they experience diseases of longevity such as cardiovascular disease and diabetes mellitus. They are also exposed to similar risk factors for ischaemic stroke, including obesity, hypertension and environmental exposures such as pollution and passive smoking.
Histopathological reports of ischaemic stroke in dogs are sparse and studies including a detailed evaluation of morphological changes of neurons, microglia/macrophages, and astrocytes in combination are still lacking [14–28]. In humans, neuroglia are recognized as central components of the pathophysiology of ischaemic stroke, and especially microglia have in recent years gained attention in basic research, as these cells can exert both beneficial and detrimental effects on neurons situated in peri-infarct lesions [7, 29–32].
The aim of the present study was to report histopathological findings with an emphasis on neuroglial reactions in the infarct and adjacent (peri-infarct) areas in a canine brain with a spontaneously occurring middle cerebral artery (MCA) infarct. The translational potential of canine ischaemic stroke as a spontaneous animal model of human ischaemic stroke is discussed.
The brain of a 1 year and 7 months old healthy female mixed-breed dog, euthanized at the owner’s request and donated to the University Hospital for Companion Animals for teaching and research purposes, was used as a normal control for the development of immunohistochemical (IHC) protocols and for control sections.
The use of the canine tissues was approved by the Local Administrative and Ethics Committee, Department of Veterinary Clinical and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen (Permission number 1 N/2013).
Processing of brain tissue
The brain of the ischaemic stroke case was collected within 2 h post-mortem. The brain was fixed by immersion in 4% formaldehyde for 14 weeks and stored in 0.15 M phosphate-buffered saline (PBS) with 30% sucrose and 0.1% sodium azide (pH 7.4) for 15 months at 4 °C. The brain was cut transversally into 19 slabs of 5 mm thickness. Each individual brain slab was numbered and photographed with the rostral cut-surface pointing upwards. Meninges were removed and the slabs were divided into smaller pieces in order to fit the vibratome equipment. Each piece was embedded in agar and further divided into 24 series of 70-μm thick, free-floating sections on a vibratome (Leica VT1000 S, Leica Microsystems, Ballerup, Denmark). Sections were stored in de Olmos cryoprotectant solution containing polyvinylpyrrolidone and sucrose diluted in a mixture of ethylene glycol and PBS and stored at −12 °C until further processing.
Every 24th section was stained with a solution of 0.01% toluidine blue (TB) (Merck Millipore, Hellerup, Denmark) diluted in 0.08 M Na2HPO4∙2H2O and 0.07 M citric acid in distilled H2O , and a luxol fast blue (LFB) solution (Amplicon, Odense, Denmark) , respectively. Sections were rinsed overnight in tris-buffered saline (TBS) at pH 7.4 and then mounted on gelatine-coated glass-slides and air-dried. For TB staining, slides were placed in TB for 26 min and differentiation was subsequently performed in graded series of alcohol and cleared in xylene. For LFB staining, differentiation of the sections was started by placing the sections in series of graded alcohol, and sections were then placed in LFB solution overnight at 4 °C. Next day, differentiation was continued by placing the sections in 0.05% lithiumcarbonate for 3 min and sections were counterstained using haematoxylin and eosin (HE). Coverslipping was performed using Depex mounting medium (VWR, Herlev, Denmark).
Every 24th section of the free-floating sections was selected for IHC detection of microglial Iba1 and glial fibrillary acidic protein (GFAP) in astrocytes. Rinsing and incubation procedures were performed at room temperature, unless otherwise stated. Sections were rinsed 2 × 30 min in 0.05 M TBS, pH 7.4, and then left overnight in the same solution at 4 °C. Demasking was performed by rinsing sections 2 × 15 min in a tris-EGTA buffer (TEG) followed by heat induced epitope retrieval by heating sections in TEG in a microwave oven (Moulinex Optimo Duo, Groupe SEB, Ballerup, Denmark) for 2 × 4 min at 800 W and 1 × 10 min at 480 W or until boiling. Sections were then rinsed 30 min in TBS followed by 3 × 25 min in TBS + 1% Triton X, preincubated with 10% foetal calf serum (FCS) in TBS for 1 h and incubated for 3 days at 4 °C with one of the following primary antibodies: polyclonal rabbit anti-Iba1 (1:500, Wako-Chem, Osaka, Japan) or polyclonal rabbit anti-GFAP (1:200, Dako, Glostrup, Denmark) diluted in 10% FCS in TBS. Next, sections were rinsed 3 × 15 min in TBS, 30 min in TBS + 1% Triton-X, 15 min in TBS and blocked for endogenous peroxidase activity for 30 min in 100% methanol and 0.2% hydrogen peroxide. Rinsing was then performed 15 min in TBS and 2 × 60 min in TBS + 1% Triton-X. Sections were then incubated with EnVision™ + System-HRP (Dako) overnight at 4 °C. Next, all sections were rinsed 3 × 45 min in TBS and developed in 0.05% 3,3′-diaminobenzidine (DAB) and 0.033% hydrogen peroxide. Sections were then rinsed 2 × 30 min in TBS and 30 min in a tris-buffer. Finally, sections were mounted on gelatin-coated glass-slides, and when air-dried, counterstained with TB diluted in tris-buffer to a 3/4 solution for 16 min, dehydrated in graded alcohol, cleared in xylene and mounted with Depex (VWR).
Control for antibody specificity was performed on brain tissue sections of the control dog by substituting the primary antibody with rabbit IgG (Dako) and by omitting the primary antibody in the protocol. All sections were devoid of immunostaining.
Gross examination of the ischaemic stroke brain immediately upon removal from the skull revealed a soft and oedematous area with a diameter of approximately 20 mm, which was visible on the surface of the right cerebral hemisphere in the lateral communication of the frontal and parietal lobes. A detailed examination of the brain after fixation and sectioning into slabs revealed a swollen area protruding above the natural convex curve of the right frontal and parietal cerebral lobes with flattened gyri and narrowed sulci. This location corresponded to the affected area as visualized by MRI. The lesion measured, in medial–lateral direction up to 32 mm, in ventral-dorsal direction up to 36 mm, and in rostro-caudal direction up to 35 mm. The lesion involved neocortical grey matter and centrum semiovale white matter in the caudal part of the right frontal lobe, the right parietal lobe, the lateral and superior part of the right temporal lobe, and the most caudal part of the right hippocampus. The medial parts of the right frontal and parietal lobes towards the cerebral falx, including the cingulate gyrus, the medial parts of the superior frontal gyrus and the most medial aspects of the right temporal lobe, were spared. Likewise did the corpus callosum, basal nuclei, thalamus, brainstem, and cerebellum appear normal.
The frail texture of the infarcted areas of the right hemisphere resulted in fragmentation of the tissue when sectioned by the vibratome. The numerous fragments from each section made the exact anatomical location of the pathological changes within each section difficult to discern.
Neuropathological changes in the affected area of the dog brain corresponded well to what has previously been described for 3-day-old infarcts in humans [33, 34] and experimental murine models , and included the presence of injured neurons, reactive microgliosis and astrocytosis as well as neutrophil granulocyte and macrophage infiltrations in the peri-infarct area.
In the canine ischaemic stroke brain, reactive microglia were found in the peri-infarct. The pathological changes observed in the present study are similar to those in the murine permanent MCA occlusion experimental model [36, 37]. Microglia are known to monitor the microenvironment of the brain and to react instantly to injury by undergoing morphological and functional changes [37, 38], thus neuronal death is suspected to induce transformation to phagocytic microglia with ameboid morphology as observed closest to and in the necrotic tissue in the present study . Following the dynamic role of microglia in relation to the formation of the ischaemic lesion, microglia are the subject of a growing research interest [40, 41].
Astrocytosis was demonstrated in the cortical peri-infarct zone. Astrocytes function among other by maintaining the vascular tone changes following neuronal activity, and are capable of both secreting and absorbing neural transmitters. Immediately following injury to the brain, reactive astrocytosis develops. While a negative effect of astrocytosis by increasing infarct size has been shown , astrocytes at the same time have the potential to decrease the detrimental excitotoxicity [43, 44]. It is further known, that astrocytes in damaged tissue can induce a microglial response . Whether astrocytes are primarily beneficial in terms of recovery or only exacerbate lesion progression is thus controversial . Accordingly, this cell type should be further studied in animal models of ischaemic stroke, including the dog.
Neutrophil granulocytes were recognized based on nuclear morphology, which is a method that has previously proved reliable when evaluating TB stained sections . In the present study, infiltration of neutrophil granulocytes into the necrotic centre of the canine brain parenchyma was observed (Fig. 3). This is in accordance with previous reports from experimental studies in rats and mice, which have shown that neutrophil migration into the parenchyma of a brain affected by ischaemic stroke peaks within the first 48 h [47, 48]. However, neutrophilic reactions following ischaemic stroke are not fully understood [49–52]. In humans, neutrophilic granulocytes are known to play a potentially harmful role with regard to infarct progression [53, 54]. Consequently, neutrophils in ischaemic stroke have been studied with the aim of developing novel treatments. Investigated potential targets include inhibiting activation, recruitment, and transmigration of neutrophilic granulocytes . In humans, the proportion of leukocytes made up of neutrophils in the peripheral blood is approximately 50–70% . In contrast, neutrophils in mice only constitute around 8–24% of the peripheral blood leukocytes , while the dog, interestingly, has a peripheral blood composition highly similar to humans with neutrophils forming approximately 60–80% of the peripheral blood leukocytes . It would therefore be of interest to investigate the relationship between neutrophils and blood–brain barrier breakdown, haemorrhagic transformation, and the impact on final neurological outcome  in dogs with spontaneous ischaemic stroke.
When evaluating the dog as a potential spontaneous animal stroke model, it seems relevant whether the ischaemic stroke was caused by a local thrombus or by an embolus. In the present study, a thrombus or embolus was neither identified at necropsy nor at histological examination even though this was the suspected underlying cause. This might, however, be explained by the fact that embolus reduction in vivo as well as post-mortem in dogs usually takes place within a few hours . In the present case, however, an embolus as the underlying cause of the infarct was strongly suspected due to the presence of petechial haemorrhages indicating haemorrhagic transformation, which is typically seen with embolic infarcts in humans . In humans, the majority of ischaemic stroke events are caused by thromboembolism . Atherosclerosis, which is the most frequent type of vascular pathology associated with arterial thrombosis in humans, seems rare in dogs and is most often associated with diabetes mellitus or hypothyroidism [16, 58]. Even though the T4 and free T4 levels were low and TSH was increased in the dog reported here, there were no clinical signs of concurrent hypothyroidism and no atherosclerosis was identified on histopathology. This further support the hypothesis of an embolus having caused the ischaemic stroke in the dog investigated.
The most common subtype of ischaemic stroke in humans is MCA territory infarcts , and the majority of animal models therefore aim at mimicking this subtype . MCA occlusion is also a common subtype of spontaneous stroke in dogs , and thus offers an interesting spontaneous animal stroke model. So far, experimental studies have provided a substantial insight into the pathophysiology of ischaemic stroke, but effective neuroprotective drugs in experimental studies have failed when tested in human patients. The translational gap may, in part, be a result of the animal models not being able to mimic the complexity of the human disease appropriately . A benefit of studying the pathophysiology of spontaneous stroke in dogs is that confounding factors such as anesthesia and surgical trauma of experimental models are avoided. Further, the similarities between the basic neuroanatomy of the canine and the human brain might explain the resemblance between the clinical disease observed in dogs and in humans with regard to associated neurological deficits and final outcome .
Ischaemic stroke seems to be less common in dogs than in humans . The reasons for this remain unclear, but possible explanations could be the presence of vascular anastomoses in the canine brain, the rare occurrence of atherosclerosis in dogs  and the rapid dissolution of clots in dogs . The low incidence of ischaemic stroke in dogs poses a hindrance to a widespread use of the dog as a spontaneous animal model for human ischaemic stroke. However, as studies regarding drug development for ethical reasons cannot be carried out in dogs, dogs could never fully replace existing animal stroke models. Instead, important investigations of the pathophysiology of spontaneous ischaemic stroke in dogs may contribute to bridge the translational gap between human patients and experimental animal models.
Our results are based on investigations of a single dog brain and thus cannot stand alone. In future, they should be followed by larger comparative studies, preferably using a multicenter design, which can ensure a high number of brains and support evidence-based conclusions. It would be of interest to perform further neuropathological characterisation of the reactions of neurons and neuroglia at different post stroke time points and investigations of vascular pathology seem highly relevant. Furthermore, white matter neuropathology has previously been linked to clinical deficits in humans with ischaemic stroke . It would therefore also be of interest to investigate such white matter lesions in dogs.
foetal calf serum
glial fibrillary acidic protein
haematoxylin and eosin
luxol fast blue
middle cerebral artery
magnetic resonance imaging
thyroid stimulating hormone
BBT, HG, BF, MB, and KLL conceived the study. BBT, HG and MB were responsible for the diagnostics and treatment of the individual dog. BBT, HG, BHC, AEL, BF, and KLL were responsible for developing the protocol for preanalytical processing of brain tissue and subsequent histochemistry and immunohistochemistry. BBT and HG performed all experiments and laboratory analysis under close supervision of BF and KLL. MW and BWK were responsible for the neuropathological evaluation and gave input to selection of stainings. BBT, BHC, BF, and KLL were responsible for making the figures. BBT drafted the manuscript. All authors gave substantial input to the manuscript. All authors read and approved the final manuscript.
The study was supported by the Danish Council for Independent Research (MB and KLL; Grant number 11-106689/FTP) and Fonden til Lægevidenskabens Fremme (BF, KLL and BHC). The authors wish to thank Dennis Brok for assistance with removing the dog brain from the scull, Arne Møller and Jens Christian H. Sørensen for helping with dividing the canine brain into slabs; Line Freksen Stejlsted, Alice Lundsgaard Larsen, Dorte Lyholmer, and Signe Marie Andersen for excellent technical assistance with the IHC procedures carried out.
The authors declare that they have no competing interests.
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