Adrian Tudor Balseanu

Long-term hypothermia reduces infarct volume in aged rats after focal ischemia

UNLABELLED In aged humans, stroke is a major cause of disability for which no neuroprotective measures are available. A viable alternative to conventional drug-based neuroprotective therapies is brain/body cooling, or hypothermia. In animal studies of focal ischemia, short-term hypothermia consistently reduces infarct size. Nevertheless, efficient neuroprotection requires long-term, regulated lowering of whole body temperature. Focal cerebral ischemia was produced by reversible occlusion of the right middle cerebral artery in 17-month-old male Sprague-Dawley rats. After stroke, the aged rats were exposed for 2 days to a mixture of air and a mild inhibitor of oxidative phosphorylation, hydrogen sulfide (H(2)S), which resulted in sustained, deep hypothermia (30.8+/-0.7 degrees C). Long-term hypothermia led to a 50% reduction in infarct size with a concomitant reduction in the number of phagocytic cells. At the transcription level, hypothermia caused a reduction in the mRNA coding for caspase 12, NF-kappa B and grp78 in the peri-infarcted region, suggesting an overall decrease in the transcriptional activity related to inflammation and apoptosis. Behaviorally, hypothermia was associated with better performance on tests that require complex sensorimotor skills, in the absence of obvious neurological deficits or physiological side effects, in aged rats. CONCLUSIONS Prolonged, H(2)S-induced hypothermia is a simple and efficacious method to limit the damage inflicted by stroke in aged rats.

Effects of Granulocyte-Colony Stimulating Factor After Stroke in Aged Rats

Background and Purpose— In aged humans, stroke is a major cause of disability for which no neuroprotective measures are available. Granulocyte-colony stimulating factor (G-CSF), a member of the cytokine family of growth factors, promotes brain neurogenesis and improves functional outcome after stroke in young animals. We tested the hypothesis that G-CSF provides a restorative therapeutic benefit in aged animals. Methods— Focal cerebral ischemia was produced by reversible occlusion of the right middle cerebral artery in 19- to 20-month-old male Sprague-Dawley rats. One hour after reperfusion, the aged rats were treated daily with 15 &mgr;g/kg G-CSF and for 15 days total. Rats were behaviorally tested and the brains removed for analysis at 28 days poststroke. Results— G-CSF treatment after stroke exerted a robust and sustained beneficial effect on survival rate and running function. Transient improvement after G-CSF treatment could be observed for coordinative motor function on the inclined plane test and for working memory in the radial-arm maze test. At the cellular level, G-CSF treatment increased the number of proliferating cells in the subventricular zone and dentate gyrus and also increased the number of newborn neurons in the subventricular zone ipsilateral to the lesion. Conclusions— These results suggest that G-CSF treatment in aged rats has a survival-enhancing capacity and a beneficial effect on functional outcome, most likely through supportive cellular processes such as neurogenesis.

Prolonged gaseous hypothermia prevents the upregulation of phagocytosis-specific protein Annexin 1 and causes low-amplitude EEG activity in the aged rat brain after cerebral ischemia

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. In the present study using magnetic resonance imaging, electroencephalogram recording, DNA arrays, reverse transcriptase polymerase chain reaction, western blotting and immunofluorescence, we characterized the central nervous system response to H2S-induced hypothermia and report, for the first time, that annexin A1, a major pro-inflammatory protein that is upregulated after stroke, was consistently downregulated in polymorphonuclear cells in the peri-lesional cortex of post-ischemic, aged rat brain after 48 hours of hypothermia induced by exposure to H2S. Our data suggest that long-term hypothermia may be a viable clinical approach to protecting the aged brain from cerebral injury. Our findings further suggest that, in contrast to monotherapies that have thus far uniformly failed in clinical practice, hypothermia has pleiotropic effects on brain physiology that may be necessary for effective protection of the brain after stroke.

Multimodal Approaches for Regenerative Stroke Therapies: Combination of Granulocyte Colony-Stimulating Factor with Bone Marrow Mesenchymal Stem Cells is Not Superior to G-CSF Alone

Attractive therapeutic strategies to enhance post-stroke recovery of aged brains include methods of cellular therapy that can enhance the endogenous restorative mechanisms of the injured brain. Since stroke afflicts mostly the elderly, it is highly desirable to test the efficacy of cell therapy in the microenvironment of aged brains that is generally refractory to regeneration. In particular, stem cells from the bone marrow allow an autologous transplantation approach that can be translated in the near future to the clinical practice. Such a bone marrow-derived therapy includes the grafting of stem cells as well as the delayed induction of endogenous stem cell mobilization and homing by the stem cell mobilizer granulocyte colony-stimulating factor (G-CSF). We tested the hypothesis that grafting of bone marrow-derived pre-differentiated mesenchymal cells (BM-MSCs) in G-CSF-treated animals improves the long-term functional outcome in aged rodents. To this end, G-CSF alone (50 μg/kg) or in combination with a single dose (106 cells) of rat BM MSCs was administered intravenously to Sprague-Dawley rats at 6 h after transient occlusion (90 min) of the middle cerebral artery. Infarct volume was measured by magnetic resonance imaging at 3 and 48 days post-stroke and additionally by immunhistochemistry at day 56. Functional recovery was tested during the entire post-stroke survival period of 56 days. Daily treatment for post-stroke aged rats with G-CSF led to a robust and consistent improvement of neurological function after 28 days. The combination therapy also led to robust angiogenesis in the formerly infarct core and beyond in the “islet of regeneration.” However, G-CSF + BM MSCs may not impact at all on the spatial reference-memory task or infarct volume and therefore did not further improve the post-stroke recovery. We suggest that in a real clinical practice involving older post-stroke patients, successful regenerative therapies would have to be carried out for a much longer time.

Repeated PTZ Treatment at 25-Day Intervals Leads to a Highly Efficient Accumulation of Doublecortin in the Dorsal Hippocampus of Rats

Background Neurogenesis persists throughout life in the adult mammalian brain. Because neurogenesis can only be assessed in postmortem tissue, its functional significance remains undetermined, and identifying an in vivo correlate of neurogenesis has become an important goal. By studying pentylenetetrazole-induced brain stimulation in a rat model of kindling we accidentally discovered that 25±1 days periodic stimulation of Sprague-Dawley rats led to a highly efficient increase in seizure susceptibility. Methodology/Principal Findings By EEG, RT-PCR, western blotting and immunohistochemistry, we show that repeated convulsive seizures with a periodicity of 25±1 days led to an enrichment of newly generated neurons, that were BrdU-positive in the dentate gyrus at day 25±1 post-seizure. At the same time, there was a massive increase in the number of neurons expressing the migratory marker, doublecortin, at the boundary between the granule cell layer and the polymorphic layer in the dorsal hippocampus. Some of these migrating neurons were also positive for NeuN, a marker for adult neurons. Conclusion/Significance Our results suggest that the increased susceptibility to seizure at day 25±1 post-treatment is coincident with a critical time required for newborn neurons to differentiate and integrate into the existing hippocampal network, and outlines the importance of the dorsal hippocampus for seizure-related neurogenesis. This model can be used as an in vivo correlate of neurogenesis to study basic questions related to neurogenesis and to the neurogenic mechanisms that contribute to the development of epilepsy.

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.

Neurobiology of Postischemic Recuperation in the Aged Mammalian Brain

Old age is associated with an enhanced susceptibility to stroke and poor recovery from brain injury, but the cellular processes underlying these phenomena are not well understood. Potential mechanism underlying functional recovery after brain ischemia in aged subjects include neuroinflammation, changes in brain plasticity-promoting factors, unregulated expression of neurotoxic factors, or differences in the generation of scar tissue that impedes the formation of new axons and blood vessels in the infarcted region. Studies suggest that behaviorally, aged rats were more severely impaired by ischemia than were young rats and showed diminished functional recovery. Both in old and young rats, the early intense proliferative activity following stroke leads to a precipitous formation of growthinhibiting scar tissue, a phenomenon amplified by the persistent expression of neurotoxic factors. Recent evidence shows that the human brain can respond to stroke with increased progenitor proliferation in aged patients, opening the possibilities of utilizing this intrinsic attempt for neuroregeneration of the human brain as a potential therapy for ischemic stroke.