Calpain and PARP Activation during Photoreceptor Cell Death in P23H and S334ter Rhodopsin Mutant Rats

Retinitis pigmentosa (RP) is a heterogeneous group of inherited neurodegenerative diseases affecting photoreceptors and causing blindness. Many human cases are caused by mutations in the rhodopsin gene. An important question regarding RP pathology is whether different genetic defects trigger the same or different cell death mechanisms. To answer this question, we analysed photoreceptor degeneration in P23H and S334ter transgenic rats carrying rhodopsin mutations that affect protein folding and sorting respectively. We found strong activation of calpain and poly(ADP-ribose) polymerase (PARP) in both mutants, concomitant with calpastatin down-regulation, increased oxidative DNA damage and accumulation of PAR polymers. These parameters were strictly correlated with the temporal progression of photoreceptor degeneration, mirroring earlier findings in the phosphodiesterase-6 mutant rd1 mouse, and suggesting execution of non-apoptotic cell death mechanisms. Interestingly, activation of caspases-3 and -9 and cytochrome c leakage—key events in apoptotic cell death—were observed only in the S334ter mutant, which also showed increased expression of PARP-1. The identification of the same metabolic markers triggered by different mutations in two different species suggests the existence of common cell death mechanisms, which is a major consideration for any mutation independent treatment

Zelltodmechanismen der Zapfenphotorezeptoren in Modellen mit erblicher retinaler Degeneration

In hereditary eye diseases of the Retinitis pigmentosa (RP) type, rod photoreceptors degenerate in a mutation-dependent fashion, followed by mutation independent cone cell death. Until today, no adequate treatment is available, but efforts are made to gain more knowledge about the mechanism of photoreceptor degeneration. To do so various animal models are available, showing mutations and symptoms comparable to human patients. In this study three models for RP (rd1 mouse, S334ter rat, and P23H rat) and a cone degeneration model, the cpfl1 mouse were investigated, asking whether a CRE-mediated transcription repressor, the inducible cAMP early repressor (ICER) was expressed. Since ICER is known to be involved in cell death in many neuronal and non-neural cells (Borlikova and Endo 2009), it may also have a detrimental effect on neuronal retinal cells. With immunostaining, ICER expression was observed in GCL, INL and in cone photoreceptors of all mutants and corresponding wt retinas. However, during cone degeneration or cone cell stress ICER expression was significantly upregulated in all mutant retinas. This finding suggests a role of ICER in cone degeneration and needs to be further investigated. Human vision is strongly depending on cone photoreceptors, mediating high spatial resolution and color perception. However, cone degeneration is taking place in many eye diseases such as age-related-macula-degeneration (AMD), diabetic retinopathy, achromatopsia, cone-rod-dystrophies, and RP. Furthermore, satisfactory treatment options are not available for these diseases and the detailed study of murine cone degeneration models is challenging, because rod photoreceptors outnumber cones by far. Investigations of the mechanism of cone cell death and the search for a neuroprotective compound requires a reliable, high-throughput cell based model system, which we intended to establish using the murine-photoreceptor cell line 661W (Al-Ubaidi et al. 1992). In a first step, these cells were characterized with several retinal markers, in order to find out whether these cone-like cells were suitable for the intended cone degeneration model. Thereafter, the cone degeneration of cpfl1 cones was mimicked in 661W cells by either inhibition of the cone phosphodiesterase 6, or activation of the cGMP dependent protein kinase G. Conclusively, the cpfl1-like degeneration of 661W cells may be used for further investigations of cone degeneration mechanisms and to search for neuroprotective substances.In den angeborenen Augenerkrankungen des Retinitis Pigmentosa (RP) Typs, degenerieren Mutationsabhängig zuerst die Stäbchen, während die nachfolgende Zapfendegeneration Mutationsunabhängig abläuft. Bis heute gibt es keine zufriedenstellende RP Therapie. Um eine Therapie zu finden, die möglichst gezielt in den Photorezeptor Zelltod eingreift, muss dieser genauestens erforscht und der zugrundeliegende Mechanismus bekannt sein. Hierfür gibt es diverse Mutante Tiermodelle, deren Mutationen und retinale Pathogenese denen von humanen Patienten ähnelt. In dieser Studie wurden drei RP (rd1 Maus, S334ter und P23H Ratte) und ein Zapfendegenerations-Tiermodell (cpfl1 Maus) auf Expression des zelltodfördernden Transkriptionsfaktors ICER („inducible cAMP early repressor“) untersucht. Es wurde bereits nachgewiesen, dass die Expression des Transkriptionsrepressors ICER in neuronalen und nicht-neuronalen Zellen während des Zelltodes hochreguliert ist und auch dessen zelltodfördernde Wirkung gezeigt (Borlikova and Endo 2009). Mittels immunhistochemischen Methoden konnte die ICER Expression in verschiedenen retinalen Zellen (Ganglienzellen, Zellen, der inneren Körnerschicht und Zapfen) von allen vier Photorezeptordegenerationsmodellen und in den korrespondieren Wildtypen nachgewiesen werden. Allerdings war die ICER Expression in den degenerierenden Zapfen der Mutanten Tiermodellen signifikant erhöht. Diese Hochregulierung der ICER Expression könnte auf eine mögliche Beteiligung von ICER am Zapfen Zelltod hinweisen. Die humane, visuelle Wahrnehmung ist stark Zapfenabhängig, nur durch diese wird die hohe Auflösung und Farbwahrnehmung garantiert. Zapfendegeneration findet jedoch in einer Vielzahl von Augenkrankheiten wie AMD (Altersbedingte Makuladegeneration), diabetische Retinopathie, Achromatopsie, Zapfen-Stäbchen-Dystrophien und RP statt, ohne dass eine zufriedenstellende Therapie existiert. Des Weiteren ist die Erforschung des Zapfenzelltodes anspruchsvoll, da in den gängigen, murinen Zapfendegenerationsmodellen zahlenmäßig die Zapfen den Stäbchen bei weitem unterlegen sind. Untersuchungen des Zapfenzelltodmechanismus und die Suche nach einem neuroprotektiven Wirkstoff benötigen ein möglichst spezifisches Zapfenzelltodmodell, welches wir unter Verwendung der Mausphotorezeptorzelllinie 661W (Al-Ubaidi et al. 1992) etablieren wollen. Hierfür wurde die Zellen zuerst mit verschiedenen retinalen Zellmarkern charakterisiert um herauszufinden, ob die 661W Zellen für das geplante Zapfenzelltodmodell geeignet sind. Anschließend wurde die Zapfendegeneration der cpfl1 Maus in den 661W Zellen nachgeahmt. Hierfür wurde pharmakologisch entweder die PDE6 inhibiert oder die PKG aktiviert. Die erzielten Resultate waren erfolgsversprechend und wir konnten zeigen, dass die degenerierenden 661W Zellen sich für Untersuchungen des Zapfenzelltodmechanismuns und für die Suche nach neuroprotektiven Substanzen eignen

Role of the kallikrein-kinin system in traumatic brain injury

Traumatic brain injury (TBI) is a major cause of mortality and morbidity worldwide. Despite improvements in acute intensive care, there are currently no specific therapies to ameliorate the effects of TBI. Successful therapeutic strategies for TBI should target multiple pathophysiologic mechanisms that occur at different stages of brain injury. The kallikrein-kinin system is a promising therapeutic target for TBI as it mediates key pathologic events of traumatic brain damage, such as edema formation, inflammation, and thrombosis. Selective and specific kinin receptor antagonists and inhibitors of plasma kallikrein and coagulation factor XII have been developed, and have already shown therapeutic efficacy in animal models of stroke and TBI. However, conflicting preclinical evaluation, as well as limited and inconclusive data from clinical trials in TBI, suggests that caution should be taken before transferring observations made in animals to humans. This review summarizes current evidence on the pathologic significance of the kallikrein-kinin system during TBI in animal models and, where available, the experimental findings are compared with human data

Early microvascular dysfunction in cerebral small vessel disease is not detectable on 3.0 Tesla magnetic resonance imaging: a longitudinal study in spontaneously hypertensive stroke-prone rats

Background Human cerebral small vessel disease (CSVD) has distinct histopathologic and imaging findings in its advanced stages. In spontaneously hypertensive stroke-prone rats (SHRSP), a well-established animal model of CSVD, we recently demonstrated that cerebral microangiopathy is initiated by early microvascular dysfunction leading to the breakdown of the blood–brain barrier and an activated coagulatory state resulting in capillary and arteriolar erythrocyte accumulations (stases). In the present study, we investigated whether initial microvascular dysfunction and other stages of the pathologic CSVD cascade can be detected by serial magnetic resonance imaging (MRI). Findings Fourteen SHRSP and three control (Wistar) rats (aged 26–44 weeks) were investigated biweekly by 3.0 Tesla (3 T) MRI. After perfusion, brains were stained with hematoxylin–eosin and histology was correlated with MRI data. Three SHRSP developed terminal CSVD stages including cortical, hippocampal, and striatal infarcts and macrohemorrhages, which could be detected consistently by MRI. Corresponding histology showed small vessel thromboses and increased numbers of small perivascular bleeds in the infarcted areas. However, 3 T MRI failed to visualize intravascular erythrocyte accumulations, even in those brain regions with the highest densities of affected vessels and the largest vessels affected by stases, as well as failing to detect small perivascular bleeds. Conclusion Serial MRI at a field strength of 3 T failed to detect the initial microvascular dysfunction and subsequent small perivascular bleeds in SHRSP; only terminal stages of cerebral microangiopathy were reliably detected. Further investigations at higher magnetic field strengths (7 T) using blood- and flow-sensitive sequences are currently underway

Intravital imaging in spontaneously hypertensive stroke-prone rats-a pilot study

Background There is growing evidence that endothelial failure and subsequent blood brain barrier (BBB) breakdown initiate cerebral small vessel disease (CSVD) pathology. In spontaneously hypertensive stroke-prone rats (SHRSP) endothelial damage is indicated by intraluminal accumulations of erythrocytes (erythrocyte thrombi) that are not observed with current magnetic resonance imaging techniques. Two-photon microscopy (2 PM) offers the potential for real-time direct detection of the small vasculature. Thus, within this pilot study we investigated the sensitivity of 2 PM to detect erythrocyte thrombi expressing initiating CSVD phenomena in vivo. Methods Eight SHRSP and 13 Wistar controls were used for in vivo imaging and subsequent histology with haematoxylin-eosin (HE). For 2 PM, cerebral blood vessels were labeled by fluorescent Dextran (70 kDa) applied intraorbitally. The correlation between vascular erythrocyte thrombi observed by 2 PM and HE-staining was assessed. Artificial surgical damage and parenchymal Dextran distribution were analyzed postmortem. Results Dextran was distributed within the small vessel walls and co-localized with IgG. Artificial surgical damage was comparable between SHRSP and Wistar controls and mainly affected the small vasculature. In fewer than 20% of animals there was correlation between erythrocyte thrombi as observed with 2 PM and histologically with HE. Conclusions Contrary to our initial expectations, there was little agreement between intravital 2 PM imaging and histology for the detection of erythrocyte thrombi. Two-photon microscopy is a valuable technique that complements but does not replace the value of conventional histology

Combined [18^{18}F]DPA-714 micro-positron emission tomography and autoradiography imaging of microglia activation after closed head injury in mice

Background Traumatic brain injury (TBI) is a major cause of death and disability. Neuroinflammation contributes to acute damage after TBI and modulates long-term evolution of degenerative and regenerative responses to injury. The aim of the present study was to evaluate the relationship of microglia activation to trauma severity, brain energy metabolism, and cellular reactions to injury in a mouse closed head injury model using combined in vivo PET imaging, ex vivo autoradiography, and immunohistochemistry. Methods A weight-drop closed head injury model was used to produce a mixed diffuse and focal TBI or a purely diffuse mild TBI (mTBI) in C57BL6 mice. Lesion severity was determined by evaluating histological damage and functional outcome using a standardized neuroscore (NSS), gliosis, and axonal injury by immunohistochemistry. Repeated intra-individual in vivo μPET imaging with the specific 18-kDa translocator protein (TSPO) radioligand [$^{18}$F]DPA-714 was performed on day 1, 7, and 16 and [$^{18}$F]FDG-μPET imaging for energy metabolism on days 2–5 after trauma using freshly synthesized radiotracers. Immediately after [$^{18}$F]DPA-714-μPET imaging on days 7 and 16, cellular identity of the [$^{18}$F]DPA-714 uptake was confirmed by exposing freshly cut cryosections to film autoradiography and successive immunostaining with antibodies against the microglia/macrophage marker IBA-1. Results Functional outcome correlated with focal brain lesions, gliosis, and axonal injury. [$^{18}$F]DPA-714-μPET showed increased radiotracer uptake in focal brain lesions on days 7 and 16 after TBI and correlated with reduced cerebral [$^{18}$F]FDG uptake on days 2–5, with functional outcome and number of IBA-1 positive cells on day 7. In autoradiography, [$^{18}$F]DPA-714 uptake co-localized with areas of IBA1-positive staining and correlated strongly with both NSS and the number of IBA1-positive cells, gliosis, and axonal injury. After mTBI, numbers of IBA-1 positive cells with microglial morphology increased in both brain hemispheres; however, uptake of [$^{18}$F]DPA-714 was not increased in autoradiography or in μPET imaging. Conclusions [$^{18}$F]DPA-714 uptake in μPET/autoradiography correlates with trauma severity, brain metabolic deficits, and microglia activation after closed head TBI

NOS knockout or inhibition but not disrupting PSD-95-NOS interaction protect against ischemic brain damage

Promising results have been reported in preclinical stroke target validation for pharmacological principles that disrupt the N-methyl-D-aspartate receptor-post-synaptic density protein-95-neuronal nitric oxide synthase complex. However, post-synaptic density protein-95 is also coupled to potentially neuroprotective mechanisms. As post-synaptic density protein-95 inhibitors may interfere with potentially neuroprotective mechanisms and sufficient validation has often been an issue in translating basic stroke research, we wanted to close that gap by comparing post-synaptic density protein-95 inhibitors with NOS1(-/-) mice and a NOS inhibitor. We confirm the deleterious role of NOS1 in stroke both invivo and invitro, but find three pharmacological post-synaptic density protein-95 inhibitors to be therapeutically ineffective

Bone marrow‐derived myeloid progenitors in the leptomeninges of adult mice

Although the bone marrow contains most hematopoietic activity during adulthood, hematopoietic stem and progenitor cells can be recovered from various extramedullary sites. Cells with hematopoietic progenitor properties have even been reported in the adult brain under steady‐state conditions, but their nature and localization remain insufficiently defined. Here, we describe a heterogeneous population of myeloid progenitors in the leptomeninges of adult C57BL/6 mice. This cell pool included common myeloid, granulocyte/macrophage, and megakaryocyte/erythrocyte progenitors. Accordingly, it gave rise to all major myelo‐erythroid lineages in clonogenic culture assays. Brain‐associated progenitors persisted after tissue perfusion and were partially inaccessible to intravenous antibodies, suggesting their localization behind continuous blood vessel endothelium such as the blood‐arachnoid barrier. Flt3$^{Cre}$ lineage tracing and bone marrow transplantation showed that the precursors were derived from adult hematopoietic stem cells and were most likely continuously replaced via cell trafficking. Importantly, their occurrence was tied to the immunologic state of the central nervous system (CNS) and was diminished in the context of neuroinflammation and ischemic stroke. Our findings confirm the presence of myeloid progenitors at the meningeal border of the brain and lay the foundation to unravel their possible functions in CNS surveillance and local immune cell production