British Medical Bulletin 65:235-257 (2003)
© 2003 The British Council
Neuroimaging of animal models of brain disease
Mark F Lythgoe*,
Nicola R Sibson
and
Neil G Harris
* RCS Unit of Biophysics, Institute of Child Health, University College London, London, UK
MRC Biochemical & Clinical Magnetic Resonance Unit, Department of Biochemistry, University of Oxford, Oxford, UK
Academic Neurosurgery, Centre for Brain Repair & Wolfson Brain Imaging Centre, University of Cambridge, Cambridge, UK
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Abstract
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The main aim of this review is to describe some of the many
animal models that have proved to be valuable from a neuroimaging
perspective. This paper complements other articles in this volume,
with a focus on animal models of the pathology of human brain
disorders for investigations with modern non-invasive neuroimaging
techniques. The use of animal model systems forms a fundamental
part of neuroscience research efforts to improve the prevention,
diagnosis, understanding and treatment of neurological conditions.
Without such models it would be impossible to investigate such
topics as the underlying mechanisms of neuronal cell damage
and death, or to screen compounds for possible anticonvulsant
properties. The adequacy of any one particular model depends
on the suitability of information gained during experimental
conditions. It is important, therefore, to understand the various
types of animal model available and choose an appropriate model
for the research question.
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Introduction
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This review covers a wide selection of experimental research
studies that combine animal models with neuroimaging. Some of
the topics (
e.g. stroke) have seen a great deal of imaging activity,
and others (
e.g. epilepsy or inflammatory disorders) are given
as examples of emergent neuroimaging disciplines. The purpose
is to illustrate that by selecting combinations of appropriate
imaging techniques and suitable animal models of disease, experimental
imaging can contribute greatly to elucidating the mechanisms
of human brain disease and to develop improved treatment strategies.
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Cerebral ischaemia
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The use of animal models in neuroimaging stroke research has
focused on a number of areas which include: (i) defining and
understanding the concept of the penumbra
1,2; (ii) development
of new imaging techniques for the diagnosis and prognosis of
stroke
3,4; and (iii) the investigation both of the underlying
processes that lead to cell death
5,6, and of possible therapies
for the treatment of stroke
7,8. Over the last decade, comprehensive
reviews have described the numerous possible animal models in
the investigation of cerebral ischaemia
9–12 and their
use in nuclear magnetic resonance (NMR) investigations
13. In
addition, the relevance of these animal models to human disease
has also been considered
14–16. The following sections
contain an overview of the types of procedures (based on the
widely used rat model) and model classifications used in experimental
stroke research, together with a brief account of various animal
models which highlight the possibilities for neuroimaging in
the investigation of cerebral ischaemia.
Experimental models may be broadly classified by a reduction in cerebral blood flow (CBF), as either global or focal models, which may, in turn, be permanent or reversible in nature. In 1975, Robinson and colleagues developed a model of permanent focal ischaemia in the rat, in which the middle cerebral artery (MCA) was coagulated distal to the rhinal fissure, the procedure being performed through a craniotomy17; this model has been used extensively with magnetic resonance (MR) to determine the temporal evolution of ischaemic damage18 and assess the efficacy of drug therapies19. Permanent MCA occlusion models do not permit the investigator to monitor the effects of reperfusion following the initial insult. To address this problem, experimenters have developed a number of methods for occluding and reperfusing the MCA. In 1986, Koizumi et al20 developed a new reversible focal ischaemic model. The MCA was occluded by a silicone rubber cylinder attached to a thread inserted through the internal carotid artery in Wistar rats. Re-circulation was accomplished by pulling the thread out of the artery21–23. This model has the advantage of avoiding craniotomy and has played an important MR role in investigating reversible ischaemic injury24, novel therapeutic strategies25, the relationship of CBF to metabolic disturbances26, delayed neuronal death27 and the diffusion-perfusion mismatch phenomena28. Permanent occlusion of the MCA may also be performed using the intraluminal suture approach which has been used with MR for the investigation of novel contrast mechanisms at high field29 and the relationship between CBF, the apparent diffusion coefficient (ADC) of water and hypoxia30. Models of focal oligaemia are not common, although they are finding increasing use in the investigation of the pathophysiology of penumbra, in which regions of oligaemic misery perfusion are present30. The aim of these experimental models is to produce a large focal lesion in which the CBF is moderately reduced throughout the MCA territory. The reduction in CBF is produced by MCA stenosis31, hypotension32 or partial obstruction of the MCA33. Further methods of inducing focal ischaemia which avoid a craniotomy include: (i) injection of homologous blood clot fragments34–36; (ii) systemic injection of Rose Bengal dye, followed by photochemical activation to induce focal thrombosis and cerebral ischaemia37; or (ii) a local application of endothelin-1, a vasoconstrictive agent, which produces a blood flow decrease with a half-time of 45–60 min38. An animal model of subarachnoid haemorrhage (SAH) has been developed using a modification of the intraluminal suture model, which allows perforation of the circle of Willis and subsequent production of a SAH34,39. Models of global ischaemia are usually transient, affecting wide-spread areas of the brain, leading to neuronal damage in selectively vulnerable regions. A period of 10–30 min of ischaemia in the four-vessel occlusion model in the rat produces cell changes that provide evidence of selective vulnerability and delayed neuronal damage40–44. Global ischaemia induced by unilateral occlusion of the CCA with hypoxic ventilation, produces ischaemic damage predominantly in the hemisphere ipsilateral to the occlusion and allows the region-specific responses to a global insult to be monitored45.
Following experimental studies in the late 1970s, the term ‘penumbra’ was introduced to designate a zone of brain tissue with moderate ischaemia and impaired neuronal function, with the neuronal paralysis being fully reversed upon reperfusion46–48. Currently, the term has a wider definition and is used to describe a region of ischaemic tissue peripheral to the core where viable neurons may be found, and thus may be potentially salvageable with suitable intervention49,50. Thus, the penumbra is an important target for acute stroke therapy. Since the penumbra can be considered as a temporary phase of potential viability through which ischaemic tissue progresses into infarction, the therapeutic time-window is limited, possibly to a few hours1,50; therefore, early detection is essential. Following occlusion of the MCA in the cat, sequential positron emission tomography (PET) studies demonstrated that CBF was decreased to < 30% of control, cerebral metabolic rate of oxygen consumption (CMRO2) was less diminished and oxygen extraction fraction (OEF) was increased, indicating misery perfusion. During permanent occlusion, the ischaemic penumbra advanced with time from the centre to the borders of the MCA territory (Fig. 1). If the MCA was reperfused after 30 min and OEF returned to normal by 24 h, no infarct was noted. However, if the initial increase in OEF diminished during the occlusion phase, the tissue would proceed on to irreversible damage2. Other PET studies have confirmed the presence of the penumbra during prolonged periods of misery perfusion, which further suggest a window of opportunity for therapy51. Penumbral zones can be delineated using several different imaging modalities, each of which have definitions that rely on the underlying mechanisms of that technique50. In 1990, Moseley et al52 observed the first diffusion-weighted imaging (DWI) changes in an animal model of cerebral ischaemia and this ADC reduction of tissue water diffusion was attributed to an osmotically obliged shift of extracellular water to intracellular compartments, as a result of a disruption of ion homeostasis and formation of cytotoxic oedema. Following this initial paper, the relationship of DWI to the penumbra has been the topic of many studies. Early work suggested that the region of signal intensity change in DWI corresponds closely to the region of peri-infarct acidosis, but also encompasses the area of ATP depletion (infarct core). Therefore, it was postulated that the outer margin of the DWI visible lesion corresponds with that of the penumbra22. However, this is only part of the picture, as it is now acknowledged that regions of DWI change that normalise on reperfusion may proceed on to infarction at a later time point27,53 and that the areas of so-called diffusion-perfusion mismatch need to be considered54.
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Fig. 1 Reconstructed surface view of left hemispheric oxygen extraction fraction in 3 cats after permanent or transient occlusion of the middle cerebral artery. Increased OEF indicates penumbra tissue, decreased OEF identifies irreversible tissue damage. Centrifugal conversion of penumbra into irreversible damage in permanent occlusion, reperfusion can only reverse penumbra and prevent damage as long as increased OEF indicates tissue viability. Image kindly provided by Prof. HD Heiss.
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The occurrence of transient waves of membrane depolarisation
known as
spreading depression (SD) emanating from an infarct
core has been suggested as one mechanism for the expansion of
tissue injury into the penumbral zone
55. During SD, the metabolic
rate of the tissue increases in response to the greatly enhanced
energy demands of the activated ion exchange pumps
56. In the
penumbra, flows are suppressed and, as a result, the increased
metabolic demand is not compensated by an increase in oxygen
and glucose
57. Eventually, ATP stores will be depleted, followed
by the cascade of pathophysiological events leading to tissue
infarction. Using DWI and ADC maps to monitor cell volume change
58,
and gradient-echo MRI to following apparent changes in blood
flow, both of these consequences can be imaged
59. The first
observation during focal ischaemia of transient abnormalities
on DWI, co-incident with transient depolarisations, was described
by Gyngell
et al60. The change in ADC associated with SD has
been used to study the pathological basis by which SD leads
to infarct growth and the mechanism whereby neuroprotective
drugs may have their therapeutic effect
61–63.
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Epilepsy
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The epilepsies are a group of disorders with a wide range of
presentations and multiple aetiologies
64. In order to model
such a diverse condition, several different and quite diverse
approaches have been developed, and currently no single model
is appropriate. In 1972, Purpura
et al65 produced a comprehensive
guide covering many experimental models of epilepsy used in
research, which still is an extremely useful guide to the various
methods used to induce epilepsy or seizures. Clinically, the
epilepsies are characterised by spontaneous recurrent epileptic
seizures, which are caused by generalised paroxysmal or partial
(focal) discharges in the brain
64. Various animal models have
been selected to highlight the classification of epilepsy disorders,
questions related to epileptogenic mechanisms, brain injury
associated with seizures and assessment of the efficacy of anti-epileptic
drugs.
Animal models of generalised seizures include the animals affected by genetic reflex epilepsy66, such as the baboon (Papio papio)67, mouse (DBA/2J)68, genetically epilepsy-prone rats (GEPRs)69, rabbits70, and the Fayoumi chicken71. Seizures may be induced by intermittent light stimulation, noise, movement, and stress65,66. Another animal model that exhibits generalised seizures is the maximal electroshock model which involves electrical stimulus to evoke a tonic extension of the hind-limbs. The model is predominately used to test possible therapies for primary and secondary generalised epilepsies72. Models for simple partial seizures include focal micro-application of topical convulsants such as penicillin66, bicuculline73, picrotoxin74, strychnine75, and kainic acid76 on the cerebral cortex. Acute direct repetitive electrical stimulation can lead to discharges that persist for seconds or minutes after the electrical stimuli cease77. Chronic seizure models develop following the application of metals such as alumina hydroxide, cobalt, tungsten or zinc75,78. Spontaneous recurrent seizures may appear 2 months after the injection, and persist in some cases for several years78. Models of complex partial seizures may be induced via injection of tetanus toxin into the hippocampus; seizures occur at 1–7 days post-injection and then chronically79,80. Administration of kainic acid, an excitotoxic analogue of L-glutamate, leads to spontaneous recurrent seizures and hippocampal damage even when injected systemically81,82. A now widely used animal model for the study of epileptogenesis is the kindling model. One technique for induction is regular stimulation of chronically implanted electrodes until spontaneous generalised seizure is achieved75,83. The ubiquitous pilocarpine model presents with status epilepticus usually within 1–2 h of injection, which may last for up to 12 h and then finally progresses to spontaneous recurrent seizures84,85. Animal models of status epilepticus (SE) may be produced via techniques such as the administration of chemical convulsant86–88 or electrical stimulation89,90.
Although the majority of experimental epilepsy research has been performed outside the neuroimaging field, there has been increasing interest following some early nuclear magnetic resonance (NMR) spectroscopy investigations, and more recently imaging studies using magnetic resonance. Broadly speaking, the present goal of imaging is to characterise both the metabolic derangement and brain injury associated with seizures, using functional (spectroscopy, diffusion and perfusion imaging) and structural (T1 and T2) neuroimaging. The first in vivo MR experiment in an animal model of seizures revealed a decrease in phosphocreatine, without a change in ATP levels during seizure activity91. This and several other spectroscopy studies demonstrated the feasibility of combing animal models of epilepsy and NMR92,93. Non-invasive imaging of animal models of epilepsy started in the early 1990s. A preliminary imaging study provided evidence for tissue damage detectable by MRI following kinate injection in the rat brain. Although the procedure did not induce seizures in this study, the T1-weighted strategy provided a better image contrast for the kainic acid lesion than the T2 approach, and the diffusion-weighted images showed improved contrast for oedematous tissue92,94. Another early study imaged blood flow changes via the MR blood oxygenation level dependent (BOLD) contrast in the kainic acid model of seizures in rats. Increases in blood flow were associated with minor behavioural seizure signs; but, as seizure activity progressed, signal intensity remain near control values possibly due to the increased oxygen extraction of the tissue95.
In 1993, Zhong et al96 published a important paper using diffusion-weighted MR imaging to investigate changes associated with SE. Following intraperitoneal injection of bicuculline in the rat, the ADC in the brain decreased 14–18% during seizures. No changes occurred in T1 or T2. This result demonstrated that, during a seizure, the ADC changed in a similar fashion to that reported in ischaemia3, but under different circumstances as the blood flow is increased and the ATP stores are only modestly reduced. This finding was also confirmed during flurothyl exposure, which is thought to act on the sodium channel to alter the responsiveness of the GABA and glutamate receptors, causing convulsions at high doses97 and during frontal cortical electroshock98. This ADC decrease during seizure activity leads to the provisional hypothesis that the ADC change may be due to perturbations in intracellular cytosolic streaming96. Although this study has been supported by more recent work99, there is still no complete explanation for the ADC change in stroke or epilepsy3. Magnetic resonance spectroscopic imaging (MRSI) together with T2-weighted and diffusion-weighted imaging (DWI) was first used in experimental epilepsy to investigate selective hippocampal and piriform cortex damage following kainite-induced status epilepticus in the rat. Decreased N-acetyl aspartate (NAA), increased lactate and decreased ADC were observed at 12 h with little evidence of histological and T2-weighted changes, and it was suggested that such changes may provide a diagnostic measure for evaluating risk of neuronal damage after SE93.
The long-term temporal evolution of lesion development, investigation of epileptic activity and tissue damage are well suited to the non-invasive nature of MRI. Several studies have now monitored T2, ADC and blood brain barrier (BBB) breakdown following SE85,100–102. In the piriform and entorhinal cortices, and in the amygdala area, the T2 was increased by 24 h, then progressively normalised by 5–9 days and finally increased again in the chronic phase (9 weeks; Plate XV see end of file p.). The chronic T2 increase corresponds to gliosis, and characterises the initial step leading to development of epilepsy that could result from spontaneous seizures85.
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Plate XV Comparison between MRI and histopathology 24 h, 14 days and 9 weeks after lithium-pilocarpine-induced SE at the level of the piriform cortex (PIR; scale bar = 200 µm), the entorhinal cortex (ENT; scale bar = 200 µm) and dorsal hippocampus (DH; scale bar = 1 mm). At each time, MRI and histopathology are from the same rat. The early damage (as soon as 24 h after SE) in the piriform and entorhinal cortex correlates well with the early MRI hypersignal (arrow). Conversely, hippocampal sclerosis develops progressively together with the MRI hypersignal (arrows). Image kindly provided by Dr C. Roch.
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A relatively small body of work has been undertaken in MR investigations
of animal models of epilepsy when compared with other neurological
conditions such as stroke. The published studies have established
magnetic resonance of epilepsy models as a valuable and developing
tool.
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Traumatic brain injury
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Traumatic brain injury (TBI) results in a complex, heterogeneous
pathology that varies not only with the severity of insult but
considerably in both spatial and temporal dimensions and with
regard to the location of the initial impact. A number of experimental
models have been developed to simulate brain trauma and the
most commonly used are weight-drop
103, impact acceleration
104,
fluid percussion (FP)
105,106, and cortical contusion injury
(CCI)
107 (for reviews of these models see Gennarelli
et al108 and Lighthall
et al109). Each model is designed to simulate
closely specific components of the clinical pathology so that
no single model is necessarily the best. Although the nature
of the physical force differs between the models, the resulting
acute and chronic destruction of gross tissue elements is somewhat
similar and can be varied by adjusting the physical force used
to produce the injury.
Autoradiography is the most widely used imaging modality for TBI research, particularly for mapping cerebral blood flow or cerebral metabolic rate of glucose utilisation, but it has also been used to determine calcium accumulation110 and microglial/macrophage activation or infiltration111 after injury. Time-course analysis after CCI injury details a unilateral drop in CBF lasting for days post-injury112 with a concomitant hyper- and subsequent hypo-metabolic state inferred by CMRG measurements113. Recent advances in PET scanner technology114 has enabled non-invasive studies of metabolism to image sequentially the same rat following head-trauma115. It is likely that this methodology is set to be exploited much further for TBI research in the coming years with the increasing availability of new tracers to map different receptor distributions and gene expression114.
The continual development of MRI techniques to assess physiology is particularly advantageous to TBI research since the now routine high spatial and temporal resolution that is achieved with this technique is particularly suited to study the highly heterogeneous nature of this condition. T1 and T2-weighted sequences have been employed to monitor contusion volume116–119 and brain anatomy at various stages after injury120. A model has also been proposed to map brain water content based on the signal contrast in T1-weighted images121, although this methodology has yet to gain wide acceptance by the MR community. The high sensitivity afforded by diffusion imaging has been used to detect very early decreases in the apparent diffusion coefficient (ADC) indicating cytotoxic oedema followed by a later increase indicative of a vasogenic component122–127. Not all groups have documented this initial ADC decrease128–130 and this may be related to the model used, the injury severity or, most likely, to the placement of the measurement region-of-interest. Contrast-enhanced imaging has been used to map regions of increased blood-brain barrier permeability131–133 and initial decreases in cerebral blood volume125 following injury. However, using contrast agents to map either blood volume or perfusion in regions where the BBB is permeable is not valid for quantitative analysis, and this may be reflected in this latter study that showed CBV maps were not well correlated with histological damage together with large inter- and intra-subject variability. Using the continuous arterial spin-labelling (CASL) technique to map CBF circumvents this problem since it relies on an endogenous tracer that is formed by magnetically labelling water protons flowing into the brain134. Early reductions in CBF have been reported after CCI using CASL127,135,136 together with loss of vascular reactivity135. Monitoring CBF at different times after injury in the same rat using this technique demonstrates hypoperfusion in core (Plate XVI see p.xix) and contusion boundary regions followed by a return to normal values at the chronic stage127. These findings are in good agreement with autoradiographic data112.
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Plate XVI Coronal perfusion (CASL) images of a single rat at different time-points after cortical contusion injury demonstrating the continued perfusion deficit in the core region. [Courtesy of NG Harris, MF Lythgoe, DL Thomas, JF Utting, E De Vita, S Chen, DG Gadian, and JD Pickard.]
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Diffuse axonal injury, a hallmark of TBI, is considered to be
an important determinant of functional outcome. Magnetisation
transfer (MT) imaging, a technique that generates contrast based
on the amount and ratio of free and bound water protons, has
been shown to be sensitive to axonal injury in a number of disease
states. The quantitative measure of MT imaging – magnetisation
ratio (MTR) – is significantly decreased in regions of
axonal injury after rotational acceleration injury in the pig
137,138 and in core regions after contusion injury in the rat
127. While
the good agreement between MTR abnormalities and axonal pathology
assessed by histology together with its reported high sensitivity
for detecting relatively mild injury
138 presumably indicates
continued use of this method. Future studies may be limited
to large animals since the rat contains a much lower ratio of
white:grey matter making detection of white matter pathology
difficult at best.
Sub-dural haemorrhage that is present immediately after contusion injury is highly visible on T2* images due to the susceptibility differences relative to the surrounding tissue, although this resolves with time127. While this presents few problems for echo-planar spin echo data, it will certainly produce large image distortions for gradient echo image acquisition, for example in a functional imaging paradigm. Thus early time-point functional studies may be limited to PET or autoradiographic studies.
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Parkinson's disease
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The disease is characterised by degeneration of the nigrostriatal
dopaminergic pathway between the cell bodies in the substantia
nigra and the projections in the striatum. Striatal dopamine
depletion results in progressive loss of motor control and the
characteristic symptoms of the disease. Experimental models
of Parkinson's disease fall into two major categories: (i) pharmacological,
for example reserpine or amphetamine administration to deplete
dopamine, a largely reversible treatment; and (ii) lesioning
using neurotoxins which is permanent, for example intraparenchymal
injection of 6-hydroxy-dopamine (6-OHDA) or systemic administration
of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as reviewed
by Hantraye
139. Imaging experimental models has largely been
in the domain of PET imaging with the availability of radioligands
to monitor both pre- and postsynaptic function using 1-[
18F]-fluoro-l-dopa,
[
11C]-CFT and [
11C]-raclopride to monitor dopamine turnover,
dopamine transporter densities and D2-receptor densities, respectively,
in various models with and without neural transplants
140–142.
Cerebral metabolic mapping with autoradiography has been used
to determine the acute increase and chronic decrease in brain
activity, reflecting cell activation followed by cell loss in
regions targeted by MPTP treatment in the non-human primate
143,144.
The technique has also been used to map regions responsive to
levodopa in the 6-OHDA lesioned rat
145. This methodology has
now been largely supplanted by functional MRI
146 or pharmacological
MRI techniques with activity induction by levodopa
147,148, D1,
D2 receptor agonists
149, amphetamine
150 or dopamine transporter
agonists
151. This methodology has also been used to assess neural
transplantation
152. Using more classical anatomical MRI sequences,
temporal signal changes in T
1 and T
2 have been used to map regions
of degeneration after lesioning in non-human primates
153 and
attempts have been made to correlate T
1 relaxation times with
the abnormal accumulation of iron in degenerating dopaminergic
neurones in the 6-OHDA rat model
154.
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Huntington's disease
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This inherited disease is characterised by degeneration of GABAergic
neurones localised mainly to the caudate and putamen with projections
to the globus pallidus and substantia nigra. Experimental models
are based on acute intrapallidal lesioning using GABAergic antagonists
or N-methyl-D-aspartate-selective agonists or chronic lesioning
using systemic administration of 3-nitropropionic acid
139,155.
Since the discovery of the gene mutation for the disease, new
transgenic mouse models are being developed
156. Imaging data
are largely PET-based, using markers described for Parkinson's
disease with more experimental data utilising fluorodeoxyglucose
to map regions of reduced metabolism indicative of cell loss
157,158.
Striatal excitotoxin lesions are visible with T
2-weighted protocols
159,
although at acute time-points only diffusion-weighted imaging
provides sufficient sensitivity
160.
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Multiple sclerosis
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Despite a wealth of studies, the pathogenesis of multiple sclerosis
(MS) is still not fully understood. These difficulties in understanding
MS stem partly from the highly complex nature of the lesion
(involving demyelination, re-myelination, T-cell and macrophage
recruitment, BBB breakdown and oedema) and in part from the
inaccessibility of neuropathological correlates. A primary goal
of animal studies is to determine the relationship between the
histopathology of a disease and the MRI signal changes. Experimental
allergic encephalopathy (EAE) is an autoimmune CNS disorder
that can be induced in susceptible species and strains by peripheral
sensitisation to white matter homogenate or myelin basic protein
in adjuvant
161. Models of EAE have been developed in mice, rats,
guinea pigs and non-human primates (for reviews see Gold
et al162 and Brok
et al163), and all except the murine model have
been studied extensively using MRI. However, a major problem
with these models is that few of them adequately represent all
of the features of human MS. In addition to which, the lesions
evolve spontaneously at any site within the brain, and often
exhibit varying temporal progressions. An alternative model
that has recently emerged is the delayed-type hypersensitivity
(DTH) model in the rat
164, which involves sensitisation of the
immune system to a non-CNS antigen (heat-killed BCG) previously
deposited in the brain. The DTH response exhibits a highly reproducible
temporal progression, encompassing all of the primary features
of MS lesions; T-cell and macrophage infiltration, BBB breakdown,
oedema and tissue damage, including primary demyelination. A
major advantage of this model for longitudinal MRI studies is
that the site of the lesion is precisely dictated by the location
of the intracerebral BCG injection.
Early MRI findings in the EAE models included increased T1, increased T2 and contrast enhancement165–169, thus corresponding broadly to those found most commonly in MS patients. However, only a relatively small number of studies have investigated correlations between MRI and histopathology obtained at the same time point. An increase in T2 has been found to correspond to regions of macrophage recruitment and oedema in both guinea pig170 and rat EAE models171. However, T2 changes were also associated with demyelination in the rat171, but not in the guinea pig170. In the marmoset model of EAE, increased proton density and T2 appear to be associated with regions of either perivascular cuffing, demyelination or perivascular gliosis172. The variability of these findings suggests that T2-weighted MRI alone is not a reliable method of distinguishing purely inflammatory lesions from either demyelinating or re-myelinating lesions172,173. Contrast-enhancing lesions in MS patients are generally considered to be a sensitive marker for disease activity. In both guinea pig174 and rat175 models of EAE, BBB breakdown has been found to correlate with macrophage recruitment. In contrast, in the marmoset EAE model, which arguably provides the most accurate representation of the relapsing-remitting form of MS, contrast-enhancing areas correlated solely with acute, actively demyelinating lesions173. However, recent work in the rat DTH model176 has demonstrated that axonal injury and inflammatory events occurring within a lesion are not restricted to the period of BBB breakdown and contrast enhancement. These findings suggest that MS disease progression may persist behind an intact BBB, and, consequently, contrast-enhancement may not be an accurate marker of disease activity.
Other imaging modalities have been used less frequently in animal studies, but have yielded some interesting findings. It has been suggested that directional changes in diffusion may distinguish between acute and chronic EAE lesions177, such that in acute lesions diffusion increases in all directions, probably as a consequence of oedema, whilst in chronic lesions diffusion only increases perpendicular to the main axon axis, possibly reflecting demyelination. Recent measurements of magnetisation transfer ratio (MTR) in EAE178 have suggested that decreases in MTR, which are frequently assumed to reflect demyelination in human MS, may in fact result from inflammatory related changes to white matter structure rather than myelin loss per se. In contrast, the short component of tissue water T2 may more accurately reflect myelin content178. New contrast agents have recently provided an alternative approach to imaging EAE. A recent study has shown that the use of a superparamagnetic iron oxide contrast agent enables macrophage recruitment to the CNS to be followed in vivo179. In addition, PET studies of EAE models have demonstrated that inflammatory processes such as up-regulation of nitric oxide180 and microglial activation181 can be identified using labelled agents.
Thus, correlation of MRI and histopathological findings in animal models of MS is beginning to aid our understanding of MS and interpretation of human MS data. However, the data from these models currently exhibit a number of conflicts. These apparent discrepancies may be resolved partly on the basis of interspecies differences. However, separation of the underlying histopathological events, either with simpler models of inflammation (see below) or manipulation of the available models of MS-like lesions, may enable more accurate identification of the MRI-neuropathology correlates. In addition, the integration of more MRI modalities and novel contrast agents may more easily differentiate between the underlying histopathological events.
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Acute CNS inflammation
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The inflammatory response is part of the hosts' defence to injury
and infection, but can exacerbate tissue injury when excessive
or inappropriate. It has become clear that inflammation contributes
not only to the archetypal CNS inflammatory disease, MS, but
also to a wide variety of acute neurological and chronic neurodegenerative
diseases, such as stroke, head trauma, Alzheimer's disease,
prion disease and HIV-related dementia. Despite this, little
is known of the effects of inflammatory processes within the
CNS, or their contribution to MR images of human neuropathologies.
However, recent work has begun to investigate systematically
the effects of well-defined aspects of the inflammatory process
within the CNS. The approach used is to micro-inject focally
specific mediators of inflammation, cytokines, into the rat
brain and to investigate their individual actions using multimodel
MRI. Two of the pro-inflammatory cytokines that have been most
widely associated with CNS pathologies are interleukin-1ß
(IL-1ß) and tumour necrosis factor-
(TNF-
). IL-1ß
is believed to exacerbate neuronal loss, but the mechanisms
of its action remain unresolved. Similarly, elevated cerebral
TNF-
expression appears to correlate with disease progression
in a number of neuropathologies, but the consequences of this
expression are not known.
The first MRI study of cytokine-induced CNS inflammation182 demonstrated that IL-1ß induces an increase in cerebral blood volume (CBV), shortly followed by a neutrophil-dependent breakdown of the blood-brain barrier (BBB). A subsequent reduction in the apparent diffusion coefficient (ADC) of tissue water long outlived the neutrophil-restricted leukocyte recruitment to the CNS, and neutrophil-depletion studies indicated that both this ADC reduction and the early increase in CBV were only partially-dependent on recruited neutrophils. This study suggests that exacerbation of neuronal injury may be a result of both a direct cytotoxic action of IL-1ß on neurones and secondary by-stander damage caused by recruited leukocytes. The reduction in ADC was not associated with any indicators of ischaemia, and in fact was shortly preceded by an increase in CBV. However, IL-1ß is known to be up-regulated during cerebral ischaemia, and these findings suggest that IL-1ß may initiate events that contribute to reductions in ADC that are associated with ischaemic lesions.
In contrast to IL-1ß, TNF- has recently been shown to cause an acute reduction in CBV (Fig. 2A,D) that is mediated by the vasoconstrictor endothelin (Fig. 2D)183. There are two TNF- receptors in the brain (TNFR1 and TNFR2) and selective activation of TNFR1 alone did not elicit the reduction in CBV (Fig. 2D), indicating that the CBV decrease is dependent on activation of the TNFR2 pathway. As with IL-1ß, breakdown of the BBB (Fig. 2B) with concomitant reduction in brain water ADC (Fig. 2C) were also observed, although these were delayed relative to the changes induced by IL-1ß. In contrast to IL-1ß, TNF- induces solely macrophage recruitment to the CNS183,184. Both TNF- receptor pathways were found to contribute to BBB breakdown, ADC reduction and macrophage recruitment. These results suggest that either or both of these MRI-visible effects may be linked to macrophage recruitment. Interestingly, the delayed reduction in ADC was not dependent on the early decrease in CBV, and this study identifies further factors that may contribute to reductions in tissue water ADC observed in neuropathologies.
These MRI studies of cytokine-induced CNS inflammation identify
alterations in CBV, tissue homeostasis and the BBB as potentially
adverse effects of IL-1ß and TNF-
within the brain,
which may contribute to neuropathologies. Manipulation of the
experimental models has enabled some of the underlying mechanisms
of the MRI-visible effects to be determined and has identified
potential therapeutic targets in neuropathologies associated
with expression of these cytokines.
|
Conclusions
|
|---|
The combination of appropriate imaging techniques and suitable
animal models of disease can greatly elucidate our understanding
of human brain pathologies. This experimental imaging partnership
has contributed to the development of novel imaging techniques,
to the promotion of better diagnostic and prognostic measures,
and to elucidation of the basic mechanisms of cellular injury
leading to improved therapies.
|
Acknowledgements
|
|---|
The authors acknowledge the Wellcome Trust for their support
of the work carried out at the Radiology and Physics Unit, Institute
of Child Health and Great Ormond Street Hospital, London, and
the Medical Research Council for their support of the work carried
out at the MRC Biochemical and Clinical Magnetic Resonance Unit,
Oxford. We are grateful to David Gadian, Rod Scott and Robyn
Haselfoot for their helpful comments and suggestions. The authors
also thank Prof. HD Heiss for the contribution of a figure to
this article.
|
Footnotes
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|---|
Correspondence to: Dr MF Lythgoe, RCS Unit of Biophysics, Institute
of Child Health, 30 Guilford Street, London WC1N 1EH, UK
|
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Abstract
Introduction