Is the spontaneously hypertensive stroke prone rat a pertinent model of sub cortical ischemic stroke? A systematic review

Systematic reviews

Is the spontaneously hypertensive stroke prone rat apertinent model of sub cortical ischemic stroke?A systematic review

Emma L. Bailey1, Colin Smith2, Cathie L. M. Sudlow1,3, and Joanna M. Wardlaw�1,4

The spontaneously hypertensive stroke prone rat is best

known as an inducible model of large artery stroke. Sponta-

neous strokes and stroke propensity in the spontaneously

hypertensive stroke prone rat are less well characterized;

however, could be relevant to human lacunar stroke. We

systematically reviewed the literature to assess the brain

tissue and small vessel pathology underlying the sponta-

neous strokes of the spontaneously hypertensive stroke

prone rat. We searched systematically three online databases

from 1970 to May 2010; excluded duplicates, reviews, and

articles describing the consequences of induced middle cere-

bral artery occlusion or noncerebral pathology; and recorded

data describing brain region and the vessels examined, num-

ber of animals, age, dietary salt intake, vascular and tissue

abnormalities. Among 102 relevant studies, animals sacrificed

after developing stroke-like symptoms displayed arteriolar

wall thickening, subcortical lesions, enlarged perivascular

spaces and cortical infarcts and hemorrhages. Histopathol-

ogy, proteomics and imaging studies suggested that the

changes not due simply to hypertension. There may be

susceptibility to endothelial permeability increase that pre-

cedes arteriolar wall thickening, degeneration and perivas-

cular tissue changes; systemic inflammation may also precede

cerebrovascular changes. There were very few data on venules

or tissue changes before hypertension. The spontaneously

hypertensive stroke prone rat shows similar features to human

lacunar stroke and may be a good spontaneous model of this

complex human disorder. Further studies should focus on

structural changes at early ages and genetics to identify factors

that predispose to vascular and brain damage.

Key words: animal model, blood–brain barrier, lacunar

stroke, pathology, salt loading, SHRSP, small vessel disease

Introduction

The spontaneously hypertensive rat (SHR) family was devel-

oped by selective cross breeding of out-bred Wistar Kyoto rats

(WKY) and consists of two main members: the SHR (1963)

which has elevated blood pressure (B180 mmHg systolic) but

rarely shows signs of stroke (sometimes known as the stroke-

resistant SHR – SHRSR) (1) and the spontaneously hyperten-

sive stroke prone rat (SHRSP) (1974) (2) selectively bred from

a substrain of the SHR that showed signs of stroke and

malignant hypertension (B220 mmHg systolic). The precise

mechanisms underlying propensity for stroke and stroke

resistance are unclear. The SHR family have been used to

model a wide range of conditions from attention deficit

hyperactivity disorder to osteoporosis. In stroke research, the

SHRSP has been mostly used in middle cerebral artery (MCA)

occlusion models to produce large MCA territory infarcts. In

contrast, the spontaneous strokes suffered by the SHRSP have

received less attention but are generally assumed to be con-

sequent upon elevated blood pressure and manifest as cortical

and subcortical infarcts and hemorrhages (3).

In systematic reviews of potential animal models of lacunar

stroke, it has been noted (4, 5) that the SHRSP seemed to

demonstrate similar pathological features to the small vessel

disease underlying human lacunar stroke pathology (6). Given

that human small cerebral vessels are difficult to study

and tissue for histopathology is difficult to obtain, the cause

of human lacunar stroke remains unclear (7). Numerous

studies describe the cerebral pathology encountered in

the SHRSP but no systematic summary of this literature isDOI: 10.1111/j.1747-4949.2011.00659.x

Conflict of interest: None declared.

Funding: E. L. B. is funded by a Medical Research Council PhD

studentship. J. M. W. is funded by the Scottish Funding Council through

the SINAPSE Collaboration (Scottish Imaging Network, A Platform for

Scientific Excellence, http://www.sinapse.ac.uk). C. L. M. S. is funded by

the Scottish Funding Council.

Correspondence: Joanna M. Wardlaw�, Division of Clinical

Neurosciences, University of Edinburgh, Western General Hospital,

Crewe Road, EH4 2XU Edinburgh, UK.

E-mail: [email protected] of Clinical Neurosciences, University of Edinburgh, Western

General Hospital, Edinburgh, UK2Division of Pathology, University of Edinburgh, Edinburgh, UK3Centre for Molecular Medicine, Institute of Genetics and Molecular

Medicine, University of Edinburgh, Edinburgh, UK4SINAPSE Collaboration (Scottish Imaging Network, A Platform for

Scientific Excellence), University of Edinburgh, 1 George Square, UK

& 2011 The Authors.International Journal of Stroke & 2011 World Stroke Organization Vol 6, October 2011, 434–444434

http://www.sinapse.ac.uk

mailto:[email protected]

available. Clarification of the pathology underlying the sponta-

neous vascular and tissue lesions in the SHRSP would aid under-

standing of human lacunar stroke and related small vessel disease.

Therefore, we gathered all published studies documenting

spontaneous cerebral pathological changes in both vessels and

tissues of the SHRSP for a detailed assessment of the type and

sequence of vascular and tissue damage (8).

Materials and methods

We searched using the terms ‘SHRSP’ and/or ‘spontaneously

hypertensive stroke prone rat’ and all possible variants of these

text strings in three online databases, Medline, Embase and

Biosis Previews. We limited the search to full-text primary

research articles, describing cerebral vascular or tissue pathol-

ogy published between 1974 (the SHRSP was developed in

1974) and May 2010. We removed duplicate publications,

conference abstracts, and reviews, and publications on artifi-

cially induced ischemia, noncerebral changes, drug interven-

tions and genetics. Where multiple publications arose from the

same animal cohort, we attributed all results to one study to

avoid artificially inflating total sample size.

We extracted data on:

� the pathology studied (e.g. vessels, tissue or both)

� methodologies

� the number of SHRSP and control animals

� their age

� the area of the vasculature and/or brain studied

� blood pressure and its relationship to pathological changes,

and

� diet (whether the rats were raised on a ‘Japanese diet’ (high

sodium (4%), low potassium (0�75%)), had sodium chloride

(1%) supplemented into their drinking water (salt loaded) (9)

or were fed standard rat chow).

We subcategorized studies into those that focused on blood

vessels alone, brain tissue alone, both vessels and tissue, and

those which examined other hematological and biochemical

changes (using serum, urine or cerebrospinal fluid (CSF)

biomarkers, e.g. nitric oxide concentration or platelet aggrega-

tion) (10–14). We defined small cerebral vessels as arterioles

that penetrated into the brain tissue and were o150 mm

diameter and larger arteries as arteries over 150 mm in diameter

including the basal intracranial large arteries, carotid/vertebral

arteries and other systemic large arteries. This dichotomy was

chosen with the aid of Paxinos’ ‘The Rat Brain in Stereotaxic

Coordinates’ (15).

We assessed study methodology with the CAMARADES

(Collaborative Approach to Meta Analysis and Review of

Animal Data from Experimental Studies) criteria (16), the

STARD (Standards for the Reporting of Diagnostic Accuracy

studies) statement (17) and MOOSE (The Meta-analysis Of

Observational Studies in Epidemiology) criteria (18) (http://

www.equatornetwork.org). We did not seek data on body

temperature, anesthesia or compliance with animal welfare

regulations, as surgical procedures were rare.

Results

The available literature

The initial search produced 4000 potentially relevant articles

(Fig 1). Excluding conference abstracts, reviews, duplicates

and artificially-induced ischemia left 496 papers. Of these, 132

papers described cerebral pathology, of which 30 were multiple

publications arising from the same animals, leaving 102

independent studies (see supporting information Table S1).

Of the 102 studies, 10 primarily described brain tissue

histopathology, 27 described vessel pathology, 14 addressed

both tissue and vessel changes and 51 described serum, urine or

CSF biomarkers. All studies used WKY, SHR (SHRSR) or

occasionally Sprague Dawley rats as controls, although Spra-

gue Dawleys were always used in conjunction with WKY.

Study methodology

None of the studies mentioned whether authors were blinded

to the SHRSP or control rat status. Blood pressure readings

were rarely documented. The total number of experimental

animals per study ranged from five (19) (an in-depth char-

acterization of tissue and vessel pathology) to 1278 (20),

Fig. 1 Search strategy for the retrieval of pathological papers to be included

in the review.

& 2011 The Authors.International Journal of Stroke & 2011 World Stroke Organization Vol 6, October 2011, 434–444 435

E. L. Bailey et al. Systematic reviews

http://www.equatornetwork.org

http://www.equatornetwork.org

median 43 (interquartile range 24–77). Nineteen studies

(18%) did not state the number of rats used. Age at sacrifice

(given in 97 studies) ranged from animals that were developing

hypertension (i.e. more than nine-weeks old), had established

hypertension (412 weeks old) or had reached the high-risk

age for stroke (416 weeks). Only nine studies documented

changes in prehypertensive animals (i.e. less than nine-weeks

old) (Fig. 2). Five studies did not state age of sacrifice. In 24

studies (23%), rats were fed a Japanese diet or had salt added to

their drinking water (Fig. 3). Use of salt was rare before the

1980s (Fig. 3) but increased from 1990 onwards. We indicate

whether animals were salt loaded in relevant sections of the

results. All studies that assessed inflammatory biomarkers (e.g.

thiostatin) used animals raised on high salt (via diet or water

supplements, n 5 5) or did not state the diet used (n 5 2).

Examination of tissue/vessel changes was mostly by histol-

ogy (e.g. hematoxylin and eosin), immunohistochemistry or

electron microscopy. Other methods included in vivo magnetic

resonance imaging (MRI) proteomics or combinations of

these. Cerebral blood flow was measured with laser Doppler

or hydrogen clearance methods.

Vascular pathology was described in 41 studies (27 vessels

only; 14 both tissue and vessel changes): 23 described large

artery changes (MCA, posterior cerebral artery (PCA), basilar

artery (BA) and the internal carotid artery); eight described

arterioles of 150 mm diameter or less. The remaining 10 studies

analyzed vessels of an unspecific size and/or brain region. We

found no studies of venular pathology or prehypertensive

changes in arterioles (only large arteries) as all studies describ-

ing small vessel pathology sacrificed animals after 13 weeks of

age. Tissue damage was described in 23 studies, but most

(n 5 19) described data from the cortex (predominantly

frontal cortex) with only occasional references to deep gray

matter and little information (n 5 2) on white matter.

Structural pathological findings – brain tissue (Fig. 4)

Most (12/14) studies examined animals during or after the

onset of hypertension. Tissue damage in SHRSP seemed

mostly to occur in the frontal cortex and basal ganglia (20–

22), usually within the territory of the MCA/anterior cerebral

artery, MCA/PCA borderzones (23) and the lenticulostriate

arteries (20). Cerebral pathology included cortical infarcts,

enlarged perivascular spaces (24, 25), white matter damage,

microinfarcts and microaneurysms (3, 20, 26, 27). Studies that

examined lesion distribution found most lesions occurred in

the basal ganglia (20). The earliest brain tissue abnormalities in

SHRSP were documented with MRI (28, 29). Here, animals

raised on a Japanese diet from six-weeks of age had blood–

brain barrier (BBB) failure by 10–12 weeks on intravenous

gadolinium-enhanced MRI (28) and tissue lesions consisting

of small foci of perivascular extracellular edema as shown by

abnormal apparent diffusion coefficient of water (29). Lesions

started in the caudate and putamen (30) and spread through

the subcortical region. At sacrifice, lesions were associated with

fibrinoid necrosis in the media, although the type/size of vessel

displaying this pathology was unclear (28).

White matter lesions (WML) were more often seen in

SHRSP than in SHR and increased with advancing age (31).

At 20 weeks, WML were more frequent (Po0�05) in four

separate brain regions (corpus callosum, anterior commisure,

internal capsule and caudate) in SHRSP compared with WKY

and SHR. SHR had lesions in the caudate only (31). In SHRSP

aged 424 weeks, white matter was rarefied and cystic,

particularly in the internal capsule, for example, where mye-

linated fibers are abundant (32). At 24 weeks, in areas with BBB

leakage demonstrated with Evans blue (33), the deep arterioles

showed fibrinoid necrosis and subcortical gray/white matter

showed hemorrhages, vacuoles and ‘necrotic cysts.’ By one-

year of age 75%, of SHRSP with WML on MRI had bilateral

lesions (34).

In SHRSP aged424 weeks, perivascular spaces were dilated

around perforating arterioles (25) and within BBB leakage sites

(24). In SHRSP, the number of affected segments of vessels

increased toward the arterial borderzones. In SHRSP

aged 440 weeks, microinfarcts o150 mm in diameter were

frequently encountered in the neocortex around arterioles

Fig. 2 Ages of animals used in pathological studies. Prehypertensive,

majority of animals sacrificed at nine-weeks of age or less; hypertensive,

majority of animals sacrificed between nine- and 16 weeks of age; stroke

prone, animals sacrificed at any time over 16 weeks of age; longitudinal

study, animals sacrificed at various ages over nine-weeks of age; unknown,

data unobtainable; total number of studies 5 102.

Fig. 3 Number of studies using salt loading in pathological studies by

decade, studies that stated salt loading n 5 24. Studies that did not add salt

to the rat’s diet n 5 78.


Systematic reviews E. L. Bailey et al.

showing fibrinoid necrosis and infarcts of varying ages were

present in the cortex and subcortex (32).

Structural pathological findings – vessels (Fig. 5)

Large vessels (4150 lm)

Before, or unattributed to hypertensionPublications between 1974 and 1990 reported few structural

changes in the large arteries. Ogata found no evidence of

atherosclerosis or thrombosis in the major arteries (32),

however, a later study reported the external diameter of the

carotid artery was less in SHRSP aged 4–8 weeks compared

with WKY (35). Another study found vascular smooth muscle

cell disorganization (deviation from the normal 901 orienta-

tion) in all three layers of the media in the basilar artery of

eight-week-old SHRSP compared with age-matched WKY

(36). This was not corrected by antihypertensive agents.

Following development of/directly attributable to hypertensionNumerous studies (17/40) described large vessel changes attrib-

uted to hypertension. Hypertrophic remodelling was common

in the large arteries of SHRSP over 12 weeks of age vs. WKY (37–

40) and media:lumen ratios were increased in SHRSP vs. SHR

from eight-weeks of age. In the BA, PCA and MCA, this was

attributed to increased smooth muscle cell layers in the media

(41, 42). Increasing media thickness also caused a progressive

increase in the external diameter of the MCA. Others reported a

thicker media and/or smaller internal diameter in the BA, MCA

and PCA vs. age-matched WKY (43). A scanning and transmis-

sion electron microscopy study revealed as many as 10% of all

smooth muscle cells in the MCA of SHRSP were necrotic by 24

weeks with no necrotic vessels in age-matched WKY (44).

Increased amounts of fibronectin in large vessel arterial walls

in 13-week-old SHRSP vs. WKY was attributed to activation of

smooth muscle cells in the media (45).

Small vessels (o150 lm)

Before, or unattributed to hypertensionOnly one study examined the small vessels before development

of hypertension (25). In this study, SHRSP were sacrificed

from four-weeks of age, then every four subsequent weeks. The

authors reported no small vessel changes visible with lead

citrate and uranyl acetate staining before 12 weeks of age; a full

description of changes was not given until 16 weeks.

Following development of/directly attributable to hypertensionAfter the onset of hypertension, most pathological changes

described were in the arterioles and smaller arteriesB30 mm in

diameter (46), particularly the lateral group of lenticulostriate

arteries in the basal ganglia (20), at bifurcations and anasto-

moses between subcortical and cortical vessels.

In 10-week-old SHRSP, the mean internal diameter of ACA,

MCA branches and the ACA–MCA anastomotic collateral

arterioles were significantly smaller than in WKY rats (47).

While this may be an early response to rising blood pressure, it

may also suggest the arterioles in SHRSP are constitutionally

smaller than WKY (in addition to the large arteries (35)) as

hypertension is not fully established at 10 weeks. At 16 weeks,

focal deposition of collagen and cytoplasmic necrosis were

Fig. 4 Summary of the timescale of brain tissue pathological changes and their relation to cerebral blood flow in SHRSP. CBF, cerebral blood flow; rCBF,

regional CBF; SHRSP, spontaneously hypertensive stroke prone rat; BBB, blood–brain barrier.



noted in the outer layers of the media of penetrating vessels in

SHRSP (25). This spread along the media with advancing age

resulting in widespread medial necrosis and atrophy with

eventual replacement of the medial muscle with collagen and

cell debris (25, 48).

In 24-week-old SHRSP, the internal (84mm) and external

diameters (104mm) of the third-order branches of the PCA

were smaller than WKY (127 and 140mm, respectively) at all

levels of intravascular pressure (49). There was no accompany-

ing hypertrophy, suggesting the arterioles were constitution-

ally small, although this may have been secondary to

hypertension which is well established at 24 weeks. At 28

weeks, small/medium arterioles close to cerebral lesions

showed hyaline degeneration, fibrinoid necrosis and throm-

bosis (50). The presence of thrombi within the deep arterioles

of nine-month-old SHRSP was considered a secondary event

following luminal narrowing and accumulation of fibrin-rich

material found at BBB leakage sites (25, 33). Interestingly, in

SHRSP without evidence of BBB leakage, no necrotic lesions

were observed in the gray matter and white matter appeared

normal (33). At 28 weeks, Tagami et al. (25) noted monocyte

adhesion to endothelial cells with migration through the

arteriolar wall into the media; the monocytes carried plasma

components and then degranulated in the media, leading to

further monocyte and fibrin adhesion, with eventual luminal

narrowing, occlusion and enlarged perivascular spaces. No

studies described venular changes.

Functional changes – vessels

Large vessels (4150 lm)

Before, or independent of hypertensionOne study found loss of cerebral blood flow autoregulation in

SHRSP at six-weeks (51), suggesting an impaired vasoactive

response may precede the onset of hypertension, although the

precise location and timing of this loss was not clear. We found

no reports of vasoactive changes in large vessels before

hypertension. At 12 and 52 weeks, endothelial-dependent

relaxation was markedly reduced in the basilar arteries in

response to acetyl choline or substance P in SHRSP vs. SHR

and SHRSP/SHR hybrids (52–54). This reduction was de-

scribed as not entirely due to blood pressure as comparisons

were made between animals with similar blood pressures (55).

Following development of, or directly attributable to hyper-tensionFunctional changes were attributed directly to hypertension

(56–59). In early hypertension (eight-weeks), regional cerebral

blood flow (CBF) in SHRSP was reduced to 50% of normal

(vs. WKY) in the frontal cortex, and fell further as blood pressure

increased (60). At nine-weeks, SHRSP also showed decreased

baroreceptor sensitivity and increased sympathetic nerve out-

flow vs. age-matched WKY, possibly indicating greater sensitivity

to blood pressure changes or failure of the feedback loop (61, 62).

Fig. 5 Summary of the timescale of structural and functional changes affecting both large and small vessels with increasing age. Blood pressure begins to

increase at approximately six-weeks with hypertension fully established at 12 weeks (see Fig. 4). WKY, Wistar Kyoto; SHRSP, spontaneously hypertensive

stroke prone rat; BBB, blood–brain barrier; PCA, posterior cerebral artery; MCA, middle cerebral artery; ACA, anterior cerebral artery; HRP, horse radish

peroxidase; CBF, cerebral blood flow.



However, another study found 10-week-old SHRSP showed

normal autoregulation vs. WKY despite falling CBF (63–65).

Autoregulatory ability in SHRSP was eventually lost by 13 weeks

in the MCA and PCA in areas of prominent tissue damage (63).

At one-year, large artery resistance in SHRSP was at least double

that of WKY, indicating increased arterial stiffness or impaired

vascular reactivity (66–73). Cultured vascular smooth muscle

cells sampled from various large arteries (including cerebral)

display a variety of functional changes including impaired

relaxation and alterations in sodium, calcium and hydrogen

ion transport in adult SHRSP. All of which can contribute to

impaired vasoreactivity in SHRSP.

Small vessels (o150 lm)We found no data before the development of hypertension. We

found the available data on small vessel reactivity difficult to

interpret as reports were conflicting and much could be

secondary to hypertension (74). For example, at five- to 10

weeks, the distensibility of small PCA branches was reported to

increase in SHRSP, but larger PCA branches had decreased

distensibility vs. WKY (35). Thirty-two-week-old SHRSP also

had increased passive distensibility in cerebral arterioles

in vitro (35). At one-year, the resistance and pressure in pial

arterioles was greater in SHRSPs (66), they also showed a

‘sausage string’ appearance (66). No studies described venules.

Endothelial and systemic abnormalities

Before, or unattributed to hypertensionCerebral endothelial function was impaired in SHRSP, man-

ifesting as BBB impairment documented histopathologically

(20, 25, 33, 75–77) or on imaging (28, 29). However, all studies

of inflammatory markers in SHRSP used animals with a high

salt intake. At 10 weeks of age, proteinuria (78), raised

inflammatory blood markers (Table 1) and BBB impairment

were detected in SHRSP on MRI (28). Additionally, two groups

found early cerebral vasogenic edema secondary to BBB leak

using both proteomics and MRI (29, 79, 80). Salt loading from

six-weeks resulted in an ‘atypical inflammatory condition’ in

SHRSP vs. both SHR and WKY, manifesting as an acute-phase

protein accumulation in the serum and urine (Table 1)

(80, 86). Excretion of inflammatory serum proteins in the

urine preceded cerebral edema by three- to 15 days (79).

Following development of/directly attributable tohypertensionSeveral studies showed that endothelial function deteriorated

with increasing hypertension (42, 77, 87). Thiostatin, an acute-

phase reactant, increased sharply in the urine and serum of

SHRSP on a Japanese diet plus 1% salt loading from six-weeks

of age, peaking at least four-weeks before stroke (B15 weeks)

(81). Ogata et al. found edema fluid around abnormal

perforating arterioles (88), while Tagami described monocyte

adhesion and infiltration of plasma components through

endothelial cells in 16-week-old SHRSP on an undisclosed

diet (25). In 12-week-old SHRSP injected in vivo with horse-

radish peroxidase (HRP) via a tail vein, a greater proportion of

HRP reaction product appeared around arterioles of the

hippocampus and hypothalamus (particularly in proximal

segments) vs. age-matched WKY, indicating leakage (89, 90).

Spontaneous BBB leakage detected using intravenous Evans

blue was found in 33% of SHRSP aged five- to nine-months vs.

WKY (91). Injections of albumin and trypan blue have also

demonstrated spontaneous leakage in hypertensive 32-week-

old SHRSP vs. both WKYand SHR (48). In addition, arteriolar

endothelial cells in SHRSP had disturbed electrophysiological

function of the tight junctions, which could lead to subtle

changes in BBB polarity (92). SHRSP capillaries showed

increased endothelial cell contraction which resulted in leakage

between endothelial cells (50). Interestingly, cultured endothe-

lial cells from the thoracic aorta of eight- to nine-week-old

Table 1 The inflammatory proteins and systems in SHRSP and WKY

Inflammatory marker Difference in expression/concentration between SHRSP and WKY rats

Thiostatin Increases linearly in SHRSP with duration of salt loading starting at least four-weeks before stroke. Serum level inversely correlates

with the baseline release of NO.

Apolipoprotein A-I Twice as concentrated in male SHRSP urine compared with WKY at same age.

Gc-globulin Twice as concentrated in male SHRSP urine compared with WKY at same age.

a1-antitrypsin Increases linearly with salt loading at least four-weeks before stroke in SHRSP.

Ceruloplasmin Quadruples in concentration in SHRSP after commencing a salt loaded diet.

Transferrin Concentration decreases in SHRSP by approximately 1/3 in weeks leading to brain abnormalities.

Kallikrenin-binding

protein

Increases steadily until hypertension is established (approximately 12 weeks) then starts to decline.

Transthyretin Detected in urine approximately three-weeks before ischemic brain features become visible on MRI.

PA/plasmin system u-Pa is over expressed and catalyzes proteolysis in any damaged brain area, t-Pa expression is decreased in SHRSP.

MMP2 Highly expressed in the damaged hemisphere of SHRSP. MMPs and plasmin system potentially cooperate in matrix degradation,

tissue damage & /remodelling.

Note all studies concerning inflammatory markers were conducted in the presence of salt loading or an unknown diet initiated at six-weeks of age (22, 78–

85). NO, nitric oxide; PA, plasminogen activator; MMP, matrix metalloproteinase; SHRSP, spontaneously hypertensive stroke prone rat; WKY, Wistar Kyoto

rats; MRI, magnetic resonance imaging.



SHRSP grew faster (93), but produced less than half the

amount of tissue plasminogen activator as age-matched

WKY rats (82). Unfortunately, this has not been investigated

in cerebral vessels.

Oxidative stress is increased by hypertension and increased

reactive oxygen species were found in the rostral ventrolateral

medulla of 14-week-old SHRSP (diet unknown) (83). Nitric

oxide expression was increased in areas of BBB leak and was

implicated in the development of tissue lesions in SHRSP at 16

weeks (94–101). These are but two of several studies where

differing levels of superoxide species and the availability of

nitric oxide have been implicated in the development of

hypertension and stroke.

Discussion

Primary findings

We identified 102 studies of cerebral pathology in the SHRSP.

This review, a comprehensive and systematic analysis of the

SHRSP, highlights gaps in knowledge which could be funda-

mental to understanding the causes of subcortical stroke in

patients, a point that was recognized almost immediately after

the strain was developed (20, 32). Although SHRSP suffer both

cortical infarcts and cerebral hemorrhages, the predominant

lesions are small subcortical lesions. Of note, five separate

research groups (28, 46, 48, 79, 91) have found evidence of, or

actively demonstrated, BBB leakage in SHRSP starting before

overt vessel or tissue damage, with and without added dietary

salt. We found no evidence of atheroma as a causative

mechanism in the SHRSP literature.

Early publications describe extensive histopathological in-

vestigations of tissue and vascular changes after stroke, while

more recent studies focus on plasma biomarkers, changes in

CBF and genetics. We found only one potential sequential

histopathological investigation of arteriolar and tissue changes

throughout the SHRSP’s life (25). This study sacrificed SHRSP

every four weeks, reported on electron microscopic changes in

perforatoring arterioles and documented serial vessel wall

damage. They found monocyte adhesion and invasion of the

arteriolar wall, fibrin deposition, disruption of the vessel wall

with enlargement of the perivascular space and secondary

luminal thrombosis in latter stages (25). The relationship of

these small vessel changes to stroke lesions has not been

specifically studied, despite much agreement that the small

vessels suffer the major impact of the vascular changes (46).

Discussion of the pathological data

In the SHRSP, small vessel lipohyalinosis and fibrinoid necrosis

are associated with microinfarcts, WML and hemorrhage (20).

However, the root cause of the small vessel pathology is

unclear. The general absence of infarcts in the territory of

arterioles occluded by thrombus, the late appearance of

thrombus and sparse evidence of large artery atheroma,

mitigate against a mainly thrombotic cause (102). However,

the difficulty of identifying which small arteriole was asso-

ciated with a particular vascular territory could be partly to

blame. We found vascular damage and functional changes were

more often encountered in the smaller arterioles (o150mm).

However, the lack of information regarding arteriolar integrity

before hypertension makes it difficult to ascertain whether

hypertension was the main or initial cause of at least some of

the arteriolar features. In contrast, most of the large vessel

changes (especially functional) occurred after 12 weeks of age,

suggesting they are a response to hypertension, although

evidence was conflicting, especially regarding dilatory and

resistance mechanisms. There was no information on venules,

yet venules are increasingly recognized to be important

markers of small vessel pathology in human lacunar stroke

(103, 104) and of inflammation in the brain (105–107).

Multiple experimental approaches pointed to a primary

causative role for BBB leakage and inflammation with evidence

of increased BBB permeability in the SHRSP preceding the

development of full hypertension (12 weeks) (28, 29, 33, 46, 48,

79, 80, 108, 109). Several studies demonstrated spontaneous

BBB leakage (48, 76, 91, 110) although frequency varied,

possibly due to differences in salt intake, genetic factors (76,

111), or whether BBB leakage was actually sought. The

inflammatory acute-phase reaction seen in the SHRSP on a

high salt intake was not observed in the SHR under identical

environment conditions (80). This reaction may exacerbate

vascular dysfunction by increasing BBB permeability through

alterations at the endothelial tight junctions.

The above findings suggest that the initiating cerebral

pathological event in the SHRSP could be BBB disruption

rather than vasospasm, thrombosis or ischemia. Indeed, no

vessel alterations were seen in regions with preserved BBB

function (24). This suggests that endothelial damage could

lead to the lipohyalinosis/fibrinoid necrosis observed in older

animals alongside white matter rarefaction and cysts (19).

However, further studies are needed to determine whether this

change can be attributed to high salt intake (34).

Besides BBB dysfunction, there are alternative causative

hypotheses. Some vasoactive responses and structural changes

such as vessel wall thickening (112) may all be the result of

adaptive responses to limit the effects of rising blood pressure.

Other changes, such as the loss of autoregulation at 10 weeks,

may expose the brain to elevated systemic blood pressure and

increase the damage (63, 64, 66). Falling CBF could also lead to

ischemia, which in turn causes BBB disruption and edema (34).

However, the falling CBF could represent a loss of brain tissue to

supply following brain damage from an alternative cause (113–

118). Finally there is growing evidence to suggest a genetic

predisposition to impaired vascular reactivity in young SHRSP

which would only serve to exacerbate the effects of any co-

exisiting BBB dysfunction and subsequent hypertension.

Further studies which try to establish genetic differences within

the brains of young SHRSP which could contribute to the

‘stroke-proneness’ of this strain are needed.



Methodological considerations within the SHRSPliterature

There are methodological issues within the SHRSP literature. In

particular, no studies mentioned blinding of analysis to rat

strain, meaning the pathological results reported are potentially

susceptible to opinion bias. Not all authors stated the age of

animals at sacrifice. Most animals (where age was given) were at

least 12 weeks old, by which time hypertension is fully estab-

lished. Many vessels included in histopathological investigations

were cortical rather than subcortical, probably because it is

technologically less demanding to analyze cortical vessels.

Therefore, further study of the exact sequence of pathological

differences in the small subcortical arterioles is required. Some

studies did not give the number of animals studied and none

provided an estimate of sample size. This may be because many

were undertaken as exploratory pilot studies. If this is true, it

would mean larger definitive studies are required. Recent studies

use high salt intake (119) to hasten pathological changes,

potentially masking the natural development of disease. The

Japanese diet contains, on average, 4% sodium chloride rather

than the usual 0�4% in standard rat chow (120) and accelerates

the development of hypertension in the SHRSP (84, 121); most

SHRSP on a high salt intake die before 28 weeks. Salt loading via

drinking water usually includes 1% sodium chloride. Compared

with WKY, SHRSP are said to prefer salty water (84, 122, 123).

Dietary salt modulates the blood pressure of SHR and SHRSP

but is said to have little effect on WKY (9). A higher salt intake in

the SHRSP may increase BBB permeability, increase production

of reactive oxygen species and lead to early death (124, 125).

However, as no studies provided direct comparisons between

animals randomly allocated to high salt intake or not, it is

difficult to say what components of brain or vessel pathology are

influenced by salt rather than other factors. Furthermore, high

salt is no more a risk factor for lacunar stroke than any other

stroke subtype (126). We would therefore discourage the use of

salt when assessing pathology in relation to human small vessel

disease. These studies were performed on several different

colonies of SHRSP, all with differing genetic phenotypes,

breeding programs, levels of systolic hypertension, etc. How-

ever, it is encouraging to see concordant results from several

different groups. Testing hypotheses on rats from several

colonies would help overcome problems of colony-specific

rats. Finally, the use of both in vitro and in vivo methods to

study small vessels may have contributed to contrasting results

as the environment around the vessel influences its function, so

studies of isolated cell or other in vitro preparations should be

interpreted cautiously (49).

Conclusions

This review has added weight to the case made in two recent

reviews of potential animal models of subcortical stroke (4, 5)

that the spontaneous cerebral pathology of the SHRSP (and

without dietary or surgical interventions) mimics human

subcortical stroke. However, there is missing data which needs

to be addressed in order to fully utilize this model of the human

disease. Our review, like all reviews, has some weaknesses. We

have not described renal vessels, mesenteric arteries and other

systemic vessels. The SHRSP, like humans, do not just suffer from

a cerebral disease (127). Even though our literature search was

robust, we cannot exclude having overlooked some literature.

Future studies should include profiling inflammatory mar-

kers in the SHRSP that have not been exposed to excess salt,

more quantitative assessments of biomarkers at prehyperten-

sive ages (less than nine-weeks), detailed analysis of the

differences in all levels of vessels between SHRSP and controls

and how changes in the arterioles and venules relate to the

sequence of spontaneous tissue pathological changes.

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Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Table S1. Summary of the studies included for analysis in

chronological order top to bottom. Abbreviations: n/a 5 infor-

mation unobtainable, unclear or irrelevant. BG 5 basal ganglia,

BA 5 basilar artery, MCA 5 middle cerebral artery, ACA 5 an-

terior cerebral artery, PCA 5 posterior cerebral artery, CC 5 cor-

pus callosum, AC 5 anterior commisure, IC 5 internal capsule,

CP 5 caudate and putamen, GP 5 globus pallidus, WM 5 white

matter, CB 5 cerebellum, MB 5 midbrain, HT 5 hypothalamus,

MO 5 medulla oblongata, HP 5 hippocampus, ST 5striatum.

N 5 total number of experimental animals used – in general this

equated to 50% WKY and 50% SHRSP unless otherwise stated.

$ 5 All SHRSP, # 5 SHR included as well as WKY. SHR,

spontaneously hypertensive rat; SHRSP, spontaneously hyper-

tensive stroke prone rat; WKY, Wistar Kyoto rats.

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Is the spontaneously hypertensive stroke prone rat a pertinent model of sub cortical ischemic stroke? A systematic review