Automated quantitative gait analysis in animal models of movement disorders

<p>Abstract</p> <p>Background</p> <p>Accurate and reproducible behavioral tests in animal models are of major importance in the development and evaluation of new therapies for central nervous system disease. In this study we investigated for the first time gait parameters of rat models for Parkinson's disease (PD), Huntington's disease (HD) and stroke using the Catwalk method, a novel automated gait analysis test. Static and dynamic gait parameters were measured in all animal models, and these data were compared to readouts of established behavioral tests, such as the cylinder test in the PD and stroke rats and the rotarod tests for the HD group.</p> <p>Results</p> <p>Hemiparkinsonian rats were generated by unilateral injection of the neurotoxin 6-hydroxydopamine in the striatum or in the medial forebrain bundle. For Huntington's disease, a transgenic rat model expressing a truncated huntingtin fragment with multiple CAG repeats was used. Thirdly, a stroke model was generated by a photothrombotic induced infarct in the right sensorimotor cortex. We found that multiple gait parameters were significantly altered in all three disease models compared to their respective controls. Behavioural deficits could be efficiently measured using the cylinder test in the PD and stroke animals, and in the case of the PD model, the deficits in gait essentially confirmed results obtained by the cylinder test. However, in the HD model and the stroke model the Catwalk analysis proved more sensitive than the rotarod test and also added new and more detailed information on specific gait parameters.</p> <p>Conclusion</p> <p>The automated quantitative gait analysis test may be a useful tool to study both motor impairment and recovery associated with various neurological motor disorders.</p

Bioluminescence imaging of stroke-induced endogenous neural stem cell response

Brain injury following stroke affects neurogenesis in the adult mammalian brain. However, a complete under¬standing of the origin and fate of the endogenous neural stem cells (eNSCs) in vivo is missing. Tools and technol¬ogy that allow non-invasive imaging and tracking of eNSCs in living animals will help to overcome this hurdle. In this study, we aimed to monitor eNSCs in a photothrombotic (PT) stroke model using in vivo bioluminescence imaging (BLI). In a first strategy, inducible transgenic mice expressing firefly luciferase (Fluc) in the eNSCs were generated. In animals that received stroke, an increased BLI signal originating from the infarct region was ob¬served. However, due to histological limitations, the identity and exact origin of cells contributing to the in¬creased BLI signal could not be revealed. To overcome this limitation, we developed an alternative strategy employing stereotactic injection of conditional lentiviral vectors (Cre-Flex LVs) encoding Fluc and eGFP in the subventricular zone (SVZ) of Nestin-Cre transgenic mice, thereby specifically labeling the eNSCs. Upon induction of stroke, increased eNSC proliferation resulted in a significant increase in BLI signal between 2 days and 2 weeks after stroke, decreasing after 3 months. Additionally, the BLI signal relocalized from the SVZ towards the infarct region during the 2 weeks following stroke. Histological analysis at 90 days post stroke showed that in the peri-infarct area, 36% of labeled eNSC progeny differentiated into astrocytes, while 21% differentiated into mature neu¬rons. In conclusion, we developed and validated a novel imaging technique that unequivocally demonstrates that nestin+ eNSCs originating from the SVZ respond to stroke injury by increased proliferation, migration towards the infarct region and differentiation into both astrocytes and neurons. In addition, this new approach allows non-invasive and specific monitoring of eNSCs overtime, opening perspectives for preclinical evaluation of can¬didate stroke therapeutics

Therapeutic effects of dietary intervention on neuroinflammation and brain metabolism in a rat model of photothrombotic stroke

INTRODUCTION: A possible target for stroke management is modulation of neuroinflammation. Evidence suggests that food components may exert anti-inflammatory properties and thus may reduce stroke-induced brain damage. AIM: To investigate the efficacy of a diet, containing anti-inflammatory ingredients, as treatment for focal ischemic brain damage induced by photothrombotic stroke in the somatosensory cortex of rats. RESULTS: Brain lesions were surrounded by strong astrogliosis on both day 7 and day 21 after stroke and were accompanied by a trend toward globally decreased glucose metabolism on day 7. The investigational diet applied 2 weeks before the ischemia did not affect astrocyte activation on day 7, but reduced it at day 21. The investigational diet applied immediately after the ischemia, increased astrocyte activation on day 7 and completely reversed this effect on day 21. Moreover, postischemic intervention increased glucose metabolism in somatosensory cortex ipsilateral to the lesion on day 7. CONCLUSION: This study reveals potentially beneficial effects of a diet containing elevated amounts of anti-inflammatory nutrients on the recovery from ischemic brain damage. Therefore, dietary intervention can be considered as an adjuvant therapy for recovery from this brain pathology

Molecular Imaging of a Preclinical Model of Stroke: Functional Evaluation and Stem Cell Therapy

Stroke, caused by a sudden disruption of blood supply to the brain or by a hemorrhage in or around the brain, is often accompanied by paralysis and forms a common cause of death. Although the insights in the pathophysiology of stroke are increasing, the intravenous recombinant tissue plasiminogen activator , that simplifies reperfusion of the brain tissue through trombolysis, is still the only treatment used in clinic approved by the FDA, and is limited to a small group of patients. Recently, stem cells have been considered as a potential alternative therapy for brain damage. The stimulation of endogenous stem cells and the administration of different exogenous stem cells have been investigated in both animal and clinical settings. Until now, it is still unknown if the grafted stem cells themselves contribute to the regeneration of damaged tissue or support the recruitment of endogenous neural and/ or vascular cells to the site of brain injury. Irrespective of their mode of action, efficient imaging tools are needed to follow the fate of the grafted cells in vivo as well as stem cell survival. In this thesis, we evaluated existing MRI and BLI tools and developed new PET imaging tools for efficient labeling of stem cells and their application in a photothrombotic stroke model. In addition, we sought to characterize novel potential functional targets in the same animal model.First, We performed an extensive in vitro study on the potential toxic effects and the labeling efficiency of 3 different stem cell populations (mouse embryonic stem cells, mouse mesenchymal stem cells and rat multipotent adult progenitor cells) with three different (U)SPIOs (Resovist®, Endorem®, Sinerem®). To assess the biological effect of (U)SPIOs on the cells, we performed proliferation studies and evaluated the phenotype and differentiation capacity of the cells. We showed that successful use of (U)SPIOs for MRI based visualization requires assessment of the optimal (U)SPIO for each individual (stem) cell population to ensure the most sensitive detection without associated toxicity.Next, we evaluated the specificity of MRI contrast in a PT stroke model with and without engraftment of SPIO-labeled stem cells. We monitored animals with PT stroke versus animals with PT stroke and stem cell engraftment by T2/T2*w MRI 4-8 h and 2, 4, 6/7 and 14 days after PT stroke induction. T2*w MR images showed hypointense contrast due to the accumulation of inflammatory cells and corresponding iron accumulation and glial scar formation in the border zone of the lesion, similar to what was observed for SPIO labeled cells. These results raise caution regarding the non-invasive monitoring of SPIO-labeled transplanted stem cells by MRI in models that induce a strong inflammatory response. Third, we developed a new PET reporter gene system for the brain. No adequate PET reporter system is available for CNS thus far since available tracers either do not cross the intact BBB or have high background signals. We selected human CB2 as a reporter because of its low basal expression in healthy brain and an inactive protein mutant (D80N) was chosen to avoid interference with signal transduction. As reporter probe we used the 11C-labeled CB2 ligands, [11C]GW405833 and [11C]NE40, that readily cross the BBB. Dual modality imaging LV and AAV vectors encoding both hCB2(D80N) and fLuc or eGFP were engineered and validated in cell culture. Next, hCB2(D80N) was locoregionally overexpressed in rat striatum by stereotactic injection of LV and AAV. Kinetic PET imaging revealed specific and reversible CB2 binding of [11C]GW405833 and [11C]NE40 in the transduced rat striatum. hCB2 and fLuc expression were followed until 9 months and showed similar kinetics.In the following study, we investigated whether we could use the newly developed CB2 PET tracer [11C]NE40 and a previously described CB1 radioligand [18F]MK-9470 to non-invasively visualize the involvement of both receptors in a preclinical model of stroke over time. Imaging with [18F]MK-9470 showed a strong increase in CB1 availability at 24 h and 72 h after stroke in the cortex surrounding the lesion, extending to the insular cortex at 24 h after surgery. These alterations were specific and confirmed by CB1 immunohistochemical staining. CB2 imaging with [11C]NE40 did not show any significant differences between stroke and sham operated animals although staining for CB2 revealed very minor immunoreactivity at 1 and 2 weeks after stroke in this model. These results open perspectives to study pharmacological interventions that target the endocannabinoid pathways especially involving CB1 signaling.Finally, we aimed to image the influence of stroke on endogenous neurogenesis and stem cell migration via BLI. We used white female Nestin-Cre transgenic mice that express Cre recombinase under the control of the rat nestin promoter in combination with the injection of conditional FLExSwitch_eGFP-T2A-fLuc LV in the SVZ. This allows specific labeling of neural stem cells with both fLuc (for BLI) and eGFP (for immunohistochemistry). Stroke animals showed a 4-fold increase in photon flux at 2 days after surgery and even a 6-fold increase at 1-2 weeks post surgery, in comparison to control. Moreover, a clear relocation of the BLI signal was detected in some animals 2 weeks after surgery in comparison to previous time points. Although the latter results are preliminary, the obtained data support the ability to follow the fate of endogenous stem cells in stroke via BLI.status: publishe

In times of crisis, go liquid!

SCOPUS: ed.jinfo:eu-repo/semantics/publishe

Tackling immunomonitoring in gastrointestinal cancer

Purpose of review The growing awareness that the immune system is a key player in the antitumoral response and the excellent clinical results achieved in some settings with anti-programmed cell death 1 (PD1)/programmed death ligand 1 (PDL1) and anti-cytotoxic T-lymphocyte-Associated protein 4 (CTLA4) drugs has led to the rise of immunotherapy as a supplement or an alternative to conventional cancer treatment. The high costs associated with these therapies, their significant toxicity and the need to understand and circumvent immune escape mechanisms raise the urgent need for immunological assessment of therapy response. The study of the immunological parameters before, during and after treatment is referred to as immunomonitoring. This review discusses the current knowledge of immunomonitoring markers in gastrointestinal cancers. Recent findings The last decade has seen a collaborative effort to standardize the assays performed in clinical trials to assess response to immunotherapy. Since then, multiple studies have been conducted on blood samples, biopsies and surgical specimens to determine their immunological profiles leading to the identification of several immunological markers possessing a predictive value of response to treatment. Summary Future research will focus on detangling the predictive value of immune markers in different therapeutic models, and also to develop new noninvasive means to monitor the immune response of patients. Video abstract http://links.lww.com/COON/A20.SCOPUS: re.jinfo:eu-repo/semantics/publishe

Ras in digestive oncology: From molecular biology to clinical implications

The modalities of Ras mutation detection, its role as a predictive biomarker, mechanisms of wild-type Ras activation, and the role of Ras-directed targeted therapies will be discussed mainly in colorectal cancer. RECENT FINDINGS: RAS genotype is generally considered to be highly concordant between primary colorectal tumours and metastases. However, recent data show significant discordance between primary tumours and specific metastatic sites, but also heterogeneity within primary tumours. Moreover, the mechanisms of Ras activation expand far beyond mutations through altered expression or function of physiological Ras activators and inhibitors. Accordingly, genomic signatures of Ras or epidermal growth factor receptor (EGFR) activation are being developed and are potential predictive biomarkers of response to anti-EGFR antibodies. Finally, several recent clinical trials targeting Ras or its downstream signalling with statins or Raf inhibitors have shown promising activity in chemorefractory metastatic colorectal cancer. SUMMARY: RAS mutation remains an important biomarker predicting response to anti-EGFR therapies and perhaps clinical outcomes after surgery for metastatic colorectal cancer, but new techniques including genomic signatures need to be validated to take into account the complexity of Ras activation. The importance of Ras signalling as a therapeutic target has recently been outlined by successful clinical trials with Raf inhibitors. Copyright © Lippincott Williams &amp; Wilkins