Richard B. Banati

In-vivo measurement of activated microglia in dementia

BACKGROUND Activated microglia have a key role in the brains immune response to neuronal degeneration. The transition of microglia from the normal resting state to the activated state is associated with an increased expression of receptors known as peripheral benzodiazepine binding sites, which are abundant on cells of mononuclear phagocyte lineage. We used brain imaging to study expression of these sites in healthy individuals and patients with Alzheimers disease. METHODS We studied 15 normal individuals (age 32-80 years), eight patients with Alzheimers disease, and one patient with minimal cognitive impairment. Quantitative in-vivo measurements of glial activation were obtained with positron emission tomography (PET) and carbon-11-labelled (R)-PK11195, a specific ligand for the peripheral benzodiazepine binding site. FINDINGS In normal individuals, regional [11C](R)-PK11195 binding did not significantly change with age, except in the thalamus, where an age-dependent increase was found. By contrast, patients with Alzheimers disease showed significantly increased regional [11C](R)-PK11195 binding in the entorhinal, temporoparietal, and cingulate cortex. INTERPRETATION In-vivo detection of increased [11C](R)-PK11195 binding in Alzheimer-type dementia, including mild and early forms, suggests that microglial activation is an early event in the pathogenesis of the disease.

Visualising microglial activation in vivo.

In health, microglia reside as quiescent guardian cells ubiquitously, but isolated without any cell‐cell contacts amongst themselves, throughout the normal CNS. In disease, however, they act as swift “sensors” for pathological events, including subtle ones without any obvious structural damage. Once activated, microglia show a territorially highly restricted involvement in the disease process. This property, peculiar to microglia, confers to them diagnostic value for the accurate spatial localisation of any active disease process, acute or chronic. In the brain, the isoquinoline PK11195, a ligand for the peripheral benzodiazepine binding site (PBBS), binds with relative cellular selectivity to activated, but not resting, microglia. Labelled with carbon‐11, (R)‐PK11195 and positron emission tomography (PET) have been used for the study of inflammatory and neurodegenerative brain disease in vivo. These studies demonstrate meaningfully distributed patterns of regional [11C](R)‐PK11195 signal increases that correlate with clinically observed loss of function. Increased [11C](R)‐PK11195 binding closely mirrors the histologically well‐described activation of microglia in the penumbra of focal lesions, as well as in the distant, anterograde, and retrograde projection areas of the lesioned neural pathway. There is also some indication that in long‐standing alterations of a neural network with persistent abnormal input, additional signals of glial activation may also emerge in transsynaptic areas. These data suggest that the injured brain is less static than commonly thought and shows subtle glial responses even in macroanatomically stable appearing regions. This implies that glial activation is not solely a sign of tissue destruction, but possibly of disease‐induced adaptation or plasticity as well. Whilst further technological and methodological advances are necessary to achieve routine clinical value and feasibility, a systematic attempt to image glial cells in vivo is likely to furnish valuable information on the cellular pathology of CNS diseases and their progression within the distributed neural architecture of the brain. GLIA 40:206–217, 2002.

Small animal SPECT and its place in the matrix of molecular imaging technologies

Molecular imaging refers to the use of non-invasive imaging techniques to detect signals that originate from molecules, often in the form of an injected tracer, and observe their interaction with a specific cellular target in vivo. Differences in the underlying physical principles of these measurement techniques determine the sensitivity, specificity and length of possible observation of the signal, characteristics that have to be traded off according to the biological question under study. Here, we describe the specific characteristics of single photon emission computed tomography (SPECT) relative to other molecular imaging technologies. SPECT is based on the tracer principle and external radiation detection. It is capable of measuring the biodistribution of minute (<10(-10) molar) concentrations of radio-labelled biomolecules in vivo with sub-millimetre resolution and quantifying the molecular kinetic processes in which they participate. Like some other imaging techniques, SPECT was originally developed for human use and was subsequently adapted for imaging small laboratory animals at high spatial resolution for basic and translational research. Its unique capabilities include (i) the ability to image endogenous ligands such as peptides and antibodies due to the relative ease of labelling these molecules with technetium or iodine, (ii) the ability to measure relatively slow kinetic processes (compared with positron emission tomography, for example) due to the long half-life of the commonly used isotopes and (iii) the ability to probe two or more molecular pathways simultaneously by detecting isotopes with different emission energies. In this paper, we review the technology developments and design tradeoffs that led to the current state-of-the-art in SPECT small animal scanning and describe the position SPECT occupies within the matrix of molecular imaging technologies.

PK (‘peripheral benzodiazepine’) – binding sites in the CNS indicate early and discrete brain lesions: microautoradiographic detection of [3H]PK 11195 binding to activated microglia

The isoquinoline PK 11195 has been suggested as a marker of glial pathology in the lesioned brain. The aim of the present study is to clarify the precise cellular location of its binding site in the central nervous system. Here, we report that in the facial nucleus after facial nerve axotomy–a lesion causing a retrograde neuronal reaction without nerve cell death while keeping the blood–brain barrier intact–activated microglia are the predominant source of lesion-induced increases of PK 11195 binding. Likewise, increased PK 11195 binding is seen in the gracile nucleus after anterograde neuronal injury following sciatic nerve transection. The peak of PK 11195 binding, using the single isomer R-PK 11195, was observed 4 days after the peripheral nerve lesion, consistent with the well-known time course of microglial activation. Photoemulsion microautoradiography confirmed the restriction of PK 11195 binding to activated microglia. The increase of PK 11195 binding in the facial nucleus seen after selective cell death of facial motoneurons by retrograde suicide transport of toxic ricin, a lesion that is accompanied by the rapid transformation of microglia into phagocytes, was no higher than that seen following axotomy. This suggests that the full transformation of microglia into parenchymal phagocytes is not necessary to reach maximal levels of PK 11195 binding. PK 11195, therefore, is a well-suited marker to detect microglial activation in areas of subtle brain pathology, where neither a disturbance of the blood–brain barrier function nor the presence of macrophages and inflammatory cells indicate an on-going disease process.

Brain microglia and blood-derived macrophages: molecular profiles and functional roles in multiple sclerosis and animal models of autoimmune demyelinating disease.

Microglia and macrophages, one a brain-resident, the other a mostly hematogenous cell type, represent two related cell types involved in the brain pathology in multiple sclerosis and its autoimmune animal model, the experimental allergic encephalomyelitis. Together, they perform a variety of different functions: they are the primary sensors of brain pathology, they are rapidly recruited to sites of infection, trauma or autoimmune inflammation in experimental allergic encephalomyelitis and multiple sclerosis and they are competent presenters of antigen and interact with T cells recruited to the inflamed CNS. They also synthesise a variety of molecules, such as cytokines (TNF, interleukins), chemokines, accessory molecules (B7, CD40), complement, cell adhesion glycoproteins (integrins, selectins), reactive oxygen radicals and neurotrophins, that could exert a damaging or a protective effect on adjacent axons, myelin and oligodendrocytes. The current review will give a detailed summary on their cellular response, describe the different classes of molecules expressed and their attribution to the blood derived or brain-resident macrophages and then discuss how these molecules contribute to the neuropathology. Recent advances using chimaeric and genetically modified mice have been particularly telling about the specific, overlapping and nonoverlapping roles of macrophages and microglia in the demyelinating disease. Interestingly, they point to a crucial role of hematogenous macrophages in initiating inflammation and myelin removal, and that of microglia in checking excessive response and in the induction and maintenance of remission.

Evolution of microglial activation in patients after ischemic stroke: A [11C](R)-PK11195 PET study

We obtained [11C](R)-PK11195 PET scans in six patients at different time points between 3 and 150 days after onset of ischemic stroke in order to measure the time course of microglial activation. Increased [11C](R)-PK11195 binding around the lesion was observed as early as 3 days. Scans at later time points showed ongoing changes in the distribution of the [11C](R)-PK11195 signal, involving the area of the primary lesion and areas distant from the primary lesion site. Our data suggest that [11C](R)-PK11195 PET can be used to investigate both the primary lesion and remote pathological changes following Wallerian degeneration.

Reactive microglia in cerebral ischaemia: an early mediator of tissue damage?

Microglial cell activation is a rapidly occurring cellular response to cerebral ischaemia. Microglia proliferate, are recruited to the site of lesion, npregulate the expression of several surface molecules including major histocom‐patibility complex class I and II antigens, complement receptor and the amyloid precursor protein (APP) as well as newly expressed cytokines, e.g. interleukin‐1 and transforming growth factor pl. The ischaemia‐induced production of APP may contribute to amyloid deposition in the aged brain under conditions of hypofusion. Ultra‐structurally, microglia transform into phagocytes removing necrotic neurons but still respecting the integrity of eventually surviving neurons even in the close vicinity of necrotic neurons. Microglial activation starts within a few minutes after ischaemia and thus precedes the morphologically detectable neuronal damage. It additionally involves a transient generalized response within the first 24 hours post‐ischaemia even at sites without eventual neuronal cell death. In functional terms, the microglial reaction appears to be a double‐edged sword in ischaemia. Activated microglia may exert a cytotoxic effector function by releasing reactive oxygen species, nitric oxide, proteinases or inflammatory cytokines. All of these cytotoxic compounds may cause bystander damage following ischaemia. Pharmacological suppression of microglial activation after ischaemia has accordingly attenuated the extent of cell death and tissue damage. However, activated microglia support tissue repair by secreting factors such as transforming growth factor βl which may limit tissue damage as well as suppress astroglial scar formation. In line with ultrastructural observations microglial activation in ischaemia is a strictly controlled event. By secreting cytokines and growth factors activated microglia most likely serve seemingly opposed functions in ischaemia, i.e. maintenance as well as removal of injured neurons. Post‐ischaemic pharmacological modulation of microglial intervention in the cascade of events that lead to neuronal necrosis may help to improve the structural and functional outcome following CNS ischaemia.

Surveillance, Intervention and Cytotoxicity: Is There a Protective Role of Microglia?

The study of microglial cell biology has become the key to understanding the brains fundamental tissue reactions as well as the cellular mechanisms underlying CNS disease. This article focuses on glial-neuronal interactions with special reference to human pathology. Three important areas of brain pathology are critically reviewed: multiple sclerosis and CNS inflammation, the brain in AIDS and opportunistic infections, and neurodegenerative disorders. Although microglial cytotoxicity may cause bystander damage, e.g. in ischemia, there is little evidence to support the view that microglial activation per se is pathogenic. Results suggesting that one important normal function of microglia is to protect the integrity of the central nervous system are discussed. The concept is proposed that microglia function as a highly developed guardian to the CNS.

[11C](R)-PK11195 PET imaging of microglial activation in multiple system atrophy

Microglia, the brain’s intrinsic macrophages, bind (R)-PK11195 when activated by neuronal injury. The authors used [11C](R)-PK11195 PET to localize in vivo microglial activation in patients with multiple system atrophy (MSA). Increased [11C](R)-PK11195 binding was primarily found in the dorsolateral prefrontal cortex, putamen, pallidum, pons, and substantia nigra, reflecting the known distribution of neuropathologic changes in MSA. Providing an indicator of disease activity, [11C](R)-PK11195 PET can thus be used to characterize the in vivo neuropathology of MSA.

Microglial Reaction in the Rat Cerebral Cortex Induced by Cortical Spreading Depression

The response of microglial cells to cortical spreading depression (CSD) was studied in rat brain by immunocytochemistry. CSD was elicited for one hour by the topical application of 4M potassium chloride solution and the microglial reaction examined immunocytochemically after 4, 16, 24 and 72 hours. CSD was sufficient to induce a microglial reaction throughout the cortex at 24 hours. Activated microglial cells furthermore showed a striking de‐novo expression of major histocompatibility complex class II antigens. In contrast, no microglial reaction was observed in the cortex of sham‐operated animals. This microglial reaction in response to CSD was not associated with histologically detectable neuronal damage. These results support the view that microglial cells are extremely sensitive to changes of the brain microenvironment. Their activation may be related to changes of ion homeostasis in the brain which are not sufficient to trigger neuronal injury.