Chapter 18

■Molecular imaging is a relatively new field that aims at the visualization and follow-up of cellular dysfunction of molecular disease mechanisms in living organisms.

■Early recognition of malfunction and direct visualization of pharmacodynamics may result in optimization of therapeutic strategies and better treatment success. Furthermore, enhanced diagnosis may also have a major economic impact.

■The key element of molecular imaging is the use of biomarkers. These reporting molecules are used as probes to help image and detect particular targets or pathways.

■In ophthalmology, molecular imaging has already been successfully applied in several animal models, using both exogenous and endogenous fluorescent probes.

New diagnostic tools provide increasingly more accurate insights into disease development and pathophysiological dysfunctions, at the cellular and biochemical level. “Molecular imaging” aims at the in vivo identification of organic and cellular malfunction even before the occurrence of anatomical changes. Thus, it is expected that in addition to better knowledge of pathological mechanisms, molecular imaging will allow for earlier diagnosis, optimization of therapeutic strategies, and early assessment of success or failure of therapy (monitoring). Medicines of the future will see a major paradigm shift by molecular imaging and the introduction of molecular medicine (Fig. 18.1).

Well-established therapeutic approaches are based on the diagnosis and treatment of diseases, when symptoms or clinical signs of the disease are already present. Late recognition and delayed initiation of therapeutic measures means costly treatment and possibly a lower chance of successful treatment. For example, one great challenge of glaucoma is the fact that the definitive diagnosis can only be made when major damage has already occurred. It is estimated that visual field defects cannot be detected until there is 20–40% retinal ganglion cells loss [1]. Current diagnostic tools do not allow for in vivo visualization of damage to these key cells in glaucoma. With

regard to another major cause of visual disability: although anti-vascular endothelial growth factor (VEGF)-therapy represents a breakthrough in the treatment of age-related macular degeneration, some patients still do not benefit from the treatment or may not achieve satisfactory functional outcomes. Recent data further indicate that another group of patients with exudative AMD with initial treatment response fails to favorably respond to anti-VEGF treatment after some time [2–4]. One major disadvantage of anti-VEGF therapy as with many other current therapies is indeed the inability to actually visualize the pharmacodynamics of the drug in vivo. Does the agent actually reach its target? Does it have a sufficient treatment effect? Is the dose correct? Does the patient or is he/she developing resistance against the drug? The biological characterization of disease and their treatments in real time by molecular imaging aims to select the best treatment strategy (“theragnostics”), including optimization of dose and detection of drug resistance.

In current clinical practice imaging techniques are designed primarily for the detection of anatomical and morphological structures, while they have very low sensitivity to changes on the biological level. Notably, better insights in diseases and patients are possible by ex vivo diagnostics and histopathology based on clinical biopsy

and tissue collection. However, it is still not known how much alteration of intrinsic activity occurs as a result of tissue fixation and processing (“artifacts”). Furthermore, methods relying on postmortem analysis do not enable the effects to be monitored in real time. This also means that the same organisms or tissue cannot be studied over time.

Looking at in vivo diagnostics in ophthalmology, fluorescein angiography for example allows for functional imaging and visualization of leakage, enabling assessment of the breakdown of the retinal vascular barrier. However, the fluorescent dye Fluorescein Sodium, is a non-specific marker. This “cold” labeling does not allow for direct binding of target substances. In contrast, fundus autofluorescence imaging already represents a technology to image direct target substances by allowing for metabolic mapping of the retinal pigment epithelium [5–7], which has been made possible with the development of confocal scanning laser ophthalmoscopy with its improved sensitivity and increased image contrast [8]. However, no individual reporting molecules are detectable by fundus autofluorescence yet. Outside ophthalmology, molecular imaging technology has already been applied in patients. Using positron emission tomography (PET) and radioactive-labeled annexin A5 as apoptosis marker, programmed cell death has been visualized in vivo in heart insufficiency [9]. Six

hours after injection, the biological marker showed binding to ischemic cardiac tissue. This demonstration of molecular imaging directly in patients is impressive, although it was not possible to resolve individual cells binding the marker. Hereby, a major challenge is the investigation of interior organs with no direct optical access. In ophthalmology, the eye with its unique optical properties offers easy access to deep anatomic structures, and, thus, provides a promising organ for in vivo visualization of molecular processes in the interior of the body at high resolution.

The hand tools for molecular imaging of the eye are already present in early stages of development. Innovative technologies such as high-resolution optical coherence tomography and confocal scanning laser ophthalmoscope already allow today for real-time presentation of ocular structures with high sensitivity and high image contrast. On the other hand, growing knowledge has been gained on cellular metabolic pathways in eye diseases in recent years. Furthermore, several developments in the field of biochemistry now allow for better labeling of target substances and reporting molecules. Hereby, ­fluorescent optical approaches appear to be particularly promising for molecular imaging. When compared with the PET technique, they are not as costly and there are no concerns of radiations. The latter in particular, limits the

repeated application of PET technology in the same individual that would be important to monitor molecular processes at different time points in longitudinal studies. One other major advantage of fluorescent optical approaches is the fact that they are not limited to exogenous probes. In animal experiments, transgenic animals can express an endogenous fluorescent target molecule. These endogenous probes would be obviously limited to preclinical evaluations while exogenous probes may represent translational biomarkers, e.g., first used and investigated in animals and then translated to the clinical application. Both these approaches – endogenous and exogenous fluorescent probes – have been already used in animal experiments in ophthalmology.

In 2004, Cordeiro and co-workers were able to image individual retinal ganglion cell apoptosis in vivo in the glaucoma rat model in real time [10]. They also demonstrated the ability to visualize single nerve cell apoptosis over hours, days, and months and showing that the effects depend on the magnitude of the initial apoptotic inducer in several models of neurodegenerative disease in rat and primate. This was succeeded by injecting fluorescentlabeled annexin 5 into the vitreous and using the confocal scanning laser ophthalmoscope for identification of single apoptosing cells. This technology, which has been subsequently given the acronym DARC (detection of apoptosing retinal cells), has been used to assess drug efficacy in glaucoma and has also been shown to be useful to evaluate neuroprotective strategies [11–14]. In experimental

glaucoma, amyloid-beta deposition as a hallmark for the devastating neurodegenerative condition of Alzheimer’s disease was colocalized with apoptotic retinal ganglion cells and induced significant retinal ganglion cell apoptosis in vivo in a doseand time-dependent manner. It was further demonstrated that targeting different components of the Abeta formation and aggregation pathway can effectively reduce glaucomatous RGC apoptosis in vivo, and finally, that combining treatments (triple therapy) was more effective than monotherapy. Overall, DARC appears to be a meaningful clinical end point that is based on the direct assessment of the retinal ganglion cell death process, not only being useful in assessing treatment efficacy, but also leading to the early identification of patients with glaucoma. The next step, translation of this technology to glaucoma patients, is underway.

It is well known by numerous postmortem studies that programmed cell death in the eye is not limited to the ganglion cells and glaucoma. The process of apoptosis is implicated in disorders throughout the retina [15]. In two easy accessible models of retinal damage, laser-induced retinal damage and acute light exposure, the DARC technique has also been used to visualize apoptotic processes in the animal model beyond retinal ganglion cell death. Individual hyperfluorescent spots as ongoing retinal cell death were seen after supra-threshold laser exposure inside laser burns in vivo (Fig. 18.2), mainly in the inner nuclear layer [16]. These observations were confirmed by postmortem analysis that showed apoptotic cells in the

inner retina, while severe necrosis was observed at the level of the retinal pigment epithelium and inner choroid. In the model of acute blue light exposure over 2 h in rats, retinal flattening and the development of apoptosis within the irradiated retina occurred one day later and following dark adaptation (Fig. 18.3) [17]. Confocal live scanning through the exposed retina revealed hyperfluorescent apoptotic cells at the level of the outer retina. Histological analysis confirmed the occurrence of photoreceptor cell death and the development of cellular damage at the outer retina. Both these studies using the in vivo DARC technology confirmed previously postmortem reports of

retinal cell apoptosis. Overall, the ability to monitor changes as they occur and longitudinally as they progress promises to be a major advancement in the real-time assessment of retinal diseases and treatment effects.

As opposed to the approach with exogenous probes as applied by the DARC technology, Eter and co-workers reported in 2008 about the use of an endogenous probe. In CX(3)CR1(GFP/+) knockin mice, in which dendritic cells, macrophages, and microglia cells are constitutively fluorescent due to genetic modification, they demonstrated in vivo the inflammatory response to laser-induced damage in the fundus of the eye [18]. They revealed that