Raluca Elena Sandu

Post-stroke gaseous hypothermia increases vascular density but not neurogenesis in the ischemic penumbra of aged rats

PURPOSE In aged humans, stroke is a major cause of disability for which no neuroprotective measures are available. In animal studies of focal ischemia, short-term hypothermia often reduces infarct size. Nevertheless, efficient neuroprotection requires long-term, regulated lowering of whole body temperature. Previously, we reported that post-stroke exposure to hydrogen sulfide (H2S) effectively lowers whole body temperature and confers neuroprotection in aged animals. METHODS In the present study using behavioral tests, MRI, telemetrical EEG, BP and temperature recordings, RT-PCR and immunofluorescence, we assessed infarct size, vascular density, neurogenesis and as well as the expression of genes coding for proteasomal proteins as well as in post-stroke aged Sprague-Dawley rats exposed to H2S- induced hypothermia. RESULTS Two days exposure to mild hypothermia diminishes the expression of several genes involved in protein degradation, thereby leading to better preservation of infarcted tissue. Further, hypothermia increased the density of newly formed blood vessels in the peri-lesional cortex did not enhance neurogenesis in the infarcted area of aged rats. Likewise, there was improved recovery of fine vestibulomotor function and asymmetric sensorimotor deficit. CONCLUSION Long-term hypothermia may be a viable clinical approach by simultaneously targeting multiple processes including better tissue preservation, enhanced vascular density and improved behavioral performance.

Present Status and Future Challenges of New Therapeutic Targets in Preclinical Models of Stroke in Aged Animals with/without Comorbidities

The aging process, comorbidities, and age-associated diseases are closely dependent on each other. Cerebral ischemia impacts a wide range of systems in an age-dependent manner. However, the aging process has many facets which are influenced by the genetic background and epigenetic or environmental factors, which can explain why some people age differently than others. Therefore, there is an urgent need to identify age-related changes in body functions or structures that increase the risk for stroke and which are associated with a poor outcome. Multimodal imaging, electrophysiology, cell biology, proteomics, and transcriptomics, offer a useful approach to link structural and functional changes in the aging brain, with or without comorbidities, to post-stroke rehabilitation. This can help us to improve our knowledge about senescence firstly, and in this context, aids in elucidating the pathophysiology of age-related diseases that allows us to develop therapeutic strategies or prevent diseases. These processes, including potential therapeutical interventions, need to be studied first in relevant preclinical models using aged animals, with and without comorbidities. Therefore, preclinical research on ischemic stroke should consider age as the most important risk factor for cerebral ischemia. Furthermore, the identification of effective therapeutic strategies, corroborated with successful translational studies, will have a dramatic impact on the lives of millions of people with cerebrovascular diseases.

Increased Degradation Rates in the Components of the Mitochondrial Oxidative Phosphorylation Chain in the Cerebellum of Old Mice

Brain structures differ in the magnitude of age-related neuron loss with the cerebellum being more affected. An underlying cause could be an age-related decline in mitochondrial bioenergetics. Successful aging of mitochondria reflects a balanced turnover of proteins involved in mitochondrial biogenesis and mitophagy. Thus, an imbalance in mitochondrial turnover can contribute to the diminution of cellular function seen during aging. Mitochondrial biogenesis and mitophagy are mediated by a set of proteins including MFN1, MFN2, OPA1, DRP1, FIS1 as well as DMN1l and DNM1, all of which are required for mitochondrial fission. Using N15 labeling, we report that the turnover rates for DMN1l and FIS1 go in opposite directions in the cerebellum of 22-month-old C57BL6j mice as compared to 3-month-old mice. Previous studies have reported decreased turnover rates for the mitochondrial respiratory complexes of aged rodents. In contrast, we found increased turnover rates for mitochondrial proteins of the oxidative phosphorylation chain in the aged mice as compared to young mice. Furthermore, the turnover rate of the components that are most affected by aging –complex III components (ubiquinol cytochrome C oxidoreductase) and complex IV components (cytochrome C oxidase)– was significantly increased in the senescent cerebellum. However, the turnover rates of proteins involved in mitophagy (i.e., the proteasomal and lysosomal degradation of damaged mitochondria) were not significantly altered with age. Overall, our results suggest that an age-related imbalance in the turnover rates of proteins involved in mitochondrial biogenesis and mitophagy (DMN1l, FIS1) in conjunction with an age-related imbalance in the turnover rates of proteins of the complexes III and IV of the electron transfer chain, might impair cerebellar mitochondrial bioenergetics in old mice.

Stem cell therapies in preclinical models of stroke. Is the aged brain microenvironment refractory to cell therapy

&NA; Stroke is a devastating disease demanding vigorous search for new therapies. Initial enthusiasm to stimulate restorative processes in the ischemic brain by means of cell‐based therapies has meanwhile converted into a more balanced view recognizing impediments that may be related to unfavorable age‐associated environments. Recent results using a variety of drug, cell therapy or combination thereof suggest that, (i) treatment with Granulocyte‐Colony Stimulating Factor (G‐CSF) in aged rats has primarily a beneficial effect on functional outcome most likely via supportive cellular processes such as neurogenesis; (ii) the combination therapy, G‐CSF with mesenchymal cells (G‐CSF + BM‐MSC or G‐CSF + BM‐MNC) did not further improve behavioral indices, neurogenesis or infarct volume as compared to G‐CSF alone in aged animals; (iii) better results with regard to integration of transplanted cells in the aged rat environment have been obtained using iPS of human origin; (iv) mesenchymal cells may be used as drug carriers for the aged post‐stroke brains. Conclusion: While the middle aged brain does not seem to impair drug and cell therapies, in a real clinical practice involving older post‐stroke patients, successful regenerative therapies would have to be carried out for a much longer time. HighlightsThe aged rat brain microenvironment is not necessarily refractory to cell survival and may also support angiogenesisIt is not clear if the transplanted cells have any beneficial effect on behavioral recoveryThe efficacy of cell therapy can be enhanced by physical rehabilitationThere remain significant developmental and translational issues that remain to be resolved

Caloric restriction stabilizes body weight and accelerates behavioral recovery in aged rats after focal ischemia

Obesity and hyperinsulinemia are risk factors for stroke. We tested the hypothesis that caloric restriction, which reduces the incidence of age‐related obesity and metabolic syndrome, may represent an efficient and cost‐effective strategy for preventing stroke and its devastating consequences. To this end, we placed aged, obese Sprague‐Dawley aged rats on a calorie‐restricted diet for 8 weeks prior to the experimental infarction. Stroke in this animal model caused a progressive decrease in weight that reached a minimum at day 6 for the young rats, and at day 10 for the aged, ad libitum‐fed rats. However, in aged animals that were calorie‐restricted prior to stroke, body weight did not decrease after stroke, but we noted accelerated body weight gain shortly thereafter starting at day 5 poststroke. Moreover, calorie‐restricted aged animals showed improved behavioral recovery in tasks requiring complex sensorimotor skills, or in tasks requiring cutaneous sensitivity and sensorimotor integration or spatial memory. Likewise, calorie‐restricted aged rats showed significant poststroke increases in serum glucose, insulin, and IGF1 levels, as well as CR‐specific changes in the expression of gene transcripts involved in glycogen metabolism, IGF signaling, apoptosis, arteriogenesis, and hypoxia. In conclusion, our study shows that recovery from stroke is enhanced in aged rats by a dietary regimen that reduces body weight prior to infarct.

Co-transplantation Strategies and Combination Therapies for Stroke

Worldwide cerebrovascular disease (CVD) is increasing in parallel with modernization, changes in lifestyle, and the growing elderly population. The incidence of stroke increases significantly with age both in men and women with incidence rates accelerating above 70 years. Since stroke afflicts mostly the elderly comorbid patients, it is highly desirable to test the efficacy of cell therapies in an appropriate animal stroke model. All monotherapeutic attempts to prevent or ameliorate brain damage following stroke have failed so far. In view of previous findings indicating that stroke impacts a wide range of mechanisms, ranging from central nervous system (CNS) physiology over CNS regeneration and plasticity to the adaptive immune system in an age-dependent manner, the failure of monotherapies is perhaps not unlikely. Bone marrow-derived mesenchymal stem cells (BM MSCs) and hematopoietic stem/progenitor cells (HSPC) are the most frequently cells used in preclinical and clinical neurorestorative studies in stroke therapy. Therefore co-transplantation of BM MSCs with other cells may be a better strategy to improve microenvironment, make the grafting more efficient, and improve functional recovery after stroke. Current knowledge includes: (1) the potential for neurogenesis is also preserved in aged, stroke-injured brains; (2) the environment of the aged brain is not hostile to transplantation of BM MSC; and (3) the extent of recovery is successful in some but not all behavioral tests. However, there remain significant developmental and translational issues to be resolved in future studies such as: (1) Understanding the differentiation into specific phenotypes. Upon transplantation, the differentiated cells often de-differentiate (Kalladka and Muir Stem Cells Cloning 7:31–44, 2014). (2) Tumorigenesis remains a significant concern (Riess et al. J Neurotrauma 24(1):216–225, 2007). (3) Anti-neuroinflammatory therapies are a potential target to promote regeneration and repair in diverse injury and neurodegenerative conditions by stem cell therapy. (iv) Efficacy of cell therapy can be enhanced by physical rehabilitation (Dunnett Clin Neurol 110:43–59, 2013). We recommend that in a real clinical practice involving older poststroke patients, successful regenerative therapies would have to be carried out for a much longer time. The BM MSC therapy in aged rodents warrants further investigation including repeated administrations of therapeutic cells at several time points after stroke and using various combinations with G-CSF or other relevant growth factors/cytokines.

Cellular and Molecular Mechanisms Underlying Non-Pharmaceutical Ischemic Stroke Therapy in Aged Subjects

The incidence of ischemic stroke in humans increases exponentially above 70 years both in men and women. Comorbidities like diabetes, arterial hypertension or co-morbidity factors such as hypercholesterolemia, obesity and body fat distribution as well as fat-rich diet and physical inactivity are common in elderly persons and are associated with higher risk of stroke, increased mortality and disability. Obesity could represent a state of chronic inflammation that can be prevented to some extent by non-pharmaceutical interventions such as calorie restriction and hypothermia. Indeed, recent results suggest that H2S-induced hypothermia in aged, overweight rats could have a higher probability of success in treating stroke as compared to other monotherapies, by reducing post-stroke brain inflammation. Likewise, it was recently reported that weight reduction prior to stroke, in aged, overweight rats induced by caloric restriction, led to an early re-gain of weight and a significant improvement in recovery of complex sensorimotor skills, cutaneous sensitivity, or spatial memory. Conclusion: animal models of stroke done in young animals ignore age-associated comorbidities and may explain, at least in part, the unsuccessful bench-to-bedside translation of neuroprotective strategies for ischemic stroke in aged subjects.

Life style, Perfusion deficits and Co-morbidities Precipitate Inflammation and Cerebrovascular Disorders in Aged Subjects

Cerebrovascular diseases represent 2nd leading cause of death worldwide. Understanding how genetic predispositions and their interaction with environmental factors affect cerebrovascular diseases is fundamental for prevention, diagnosis and for the development of safe and efficient therapies. Cerebrovascular diseases have not only a very high mortality rate, but also results in debilitating neurological impairments or permanent disability in survivors associated with huge economic losses. Among the women and men individuals with a low-risk lifestyle (smoking, exercising daily, consuming a prudent diet including moderate alcohol and having a healthy weight during mid-life) had a significantly lower risk of stroke than individuals without a low-risk lifestyle. Current review focuses on determining the relationship between diet, as an important component of ‘life style’, aging and cerebrovascular diseases.This review may help to unravel biological mechanisms linking lifestyle, diet-induced, metabolic inflammation, aging and cerebral hypoperfusion to development of cerebrovascular diseases, a prerequisite for development of science-based preventive strategies needed to combat the major public health challenges like obesity and stroke.