Therapeutic hypothermia (TH) benefits survivors of cardiac arrest and neonatal hypoxic–ischemic injury and may benefit stroke patients. Large TH clinical trials, however, have shown mixed results. Given the substantial pre-clinical literature supporting TH, we explored possible mechanisms for clinical trial variability. Using a standard rodent stroke model ( n = 20 per group), we found smaller infarctions after 2 h pre- or post-reperfusion TH compared to 4 h. To explore the mechanism of this discrepancy, we used primary cell cultures of rodent neurons, astrocytes, or endothelial cells subjected to oxygen–glucose deprivation (OGD). Then, cells were randomly assigned to 33℃, 35℃ or 37℃ for varying durations after varying delay times. Both 33 and 35℃ TH effectively preserved all cell types, although 33℃ was superior. Longer cooling durations overcame moderate delays to cooling initiation. In contrast, TH interfered with astrocyte paracrine protection of neurons in a temperature-dependent manner. These findings suggest that longer TH is needed to overcome delays to TH onset, but shorter TH durations may be superior to longer, perhaps due to suppression of astrocytic paracrine support of neurons during injury. We propose a scheme for optimizing TH after cerebral injury to stimulate further studies of cardiac arrest and stroke.
Astrocytes protect neurons during cerebral injury through several postulated mechanisms. Recent therapeutic attention has focused on enhancing or augmenting the neuroprotective actions of astrocytes but in some instances astrocytes can assume a neurotoxic phenotype. The signaling mechanisms that drive astrocytes toward a protective versus toxic phenotype are not fully known but cell–cell signaling via proteases acting on cell‐specific receptors underlies critical mechanistic steps in neurodevelopment and disease. The protease activated receptor (PAR), resides in multiple brain cell types, and most PARs are found on astrocytes. We asked whether neuron‐generated thrombin constituted an important astrocyte activation signal because our previous studies have shown that neurons contain prothrombin gene and transcribed protein. We used neuron and astrocyte mono‐cell cultures exposed to oxygen‐glucose deprivation and a model of middle cerebral artery occlusion. We found that ischemic neurons secrete thrombin into culture media, which leads to astrocyte activation; such astrocyte activation can be reproduced with low doses of thrombin. Media from prothrombin‐deficient neurons failed to activate astrocytes and adding thrombin to such media restored activation. Astrocytes lacking PAR1 did not respond to neuron‐generated thrombin. Induced astrocyte activation was antagonized dose‐dependently with thrombin inhibitors or PAR1 antagonists. Ischemia‐induced astrocyte activation in vivo was inhibited after neuronal prothrombin knockout, resulting in larger strokes. Restoring prothrombin to neurons with a lentiviral gene vector restored astrocyte activation and reduced stroke damage. We conclude that neuron‐generated thrombin, released during ischemia, acts via PAR1 and may cause astrocyte activation and paracrine neuroprotection.
Objectives: Brain is a complex structure during ischemia along with neurons; components of vascular unit astrocytes, pericytes and endothelial cells play a major role in hypoxic and blood brain barrier leakage mediated injury. Thrombin acts via protease activated receptor-1 (PAR-1) and leads to neuronal survival or death, PAR-1 knockout cells survive ischemia. We hypothesize that different elements of the Brain vascular unit play different roles in responding to injury. The present study aimed to establish a correlation between astrocytes, pericytes and endothelial cells vulnerability via OGD model of ischemia and Thrombin mediated toxicity. Methods: Using highly reproducible oxygen glucose deprivation (OGD) model Primary cultured cells were exposed to OGD for various durations and Thombin doses to determine the optimum time point for 80 % cell death or cell viability measured using LDH release assay MTT assay. LD50 dose of OGD and Thrombin was used to determine the effect of various drugs mediating PAR-1 receptor pathway Results: The viability assay using MTT showed that the three elements of vascular unit respond differentially to longer durations of ischemia, with pericytes being the most vulnerable, followed by endothelial cells, followed by astrocytes. Our thrombin dose effect shows that Pericytes and endothelial respond to thrombin similarly whereas, astrocytes on lower concentration proliferate, but don’t survive the higher concentrations. Conclusion: An exciting, novel finding in our data is that different elements of the vascular unit—astrocytes, pericytes and endothelial cells—all exhibit effect of ischemia and reperfusion at different time scales. Up until recently, studies of neuroprotection addressed the brain as a homogenous unit, with the tacit assumption that all elements responded similarly. Our data on astrocytes, endothelial cells and pericytes—allows more focused investigation.
Background: Therapeutic hypothermia (TH) shows considerable promise in laboratory ischemic stroke models. Delayed therapy confounds trials of any neuroprotectant, as well as inadvertent inclusion of non-recanalized patients in most clinical trials. The advent of intra-arterial thrombectomy (IAT) offers the opportunity to combine neuroprotection with controlled, documented recanalization. In contrast to prior rodent models using surface cooling techniques, clinical TH often uses endovascular cooling technology. We modeled delayed recanalization and simulated endovascular cooling in a rodent model. Method: A perivascular catheter was implanted retroperitoneally in 8 male, 300g, Sprague Dawley rats 6 days prior to a 4h heat blunted nylon filament MCAo. At reperfusion animals were randomized by an investigator outside the laboratory to brain temperature 33°C or 37°C (n=4 per group) monitored by a temporalis muscle needle thermistor. Saline pumped through the closed loop cooling circuit convectively changed vena cava and body core temperature. After 2h the circuit was disconnected and the animals returned to cages. Bederson 3-point neuroscores were obtained 4h and 24h after occlusion. After 24 hours, rats were sacrificed for TTC exclusion. Results: Perivascular cooling achieved very rapid target temperature (< 15min) and maintained temperature within 0.5°C for 4 hours. Mean±SD neuroscores were 2.5±0.5 after 4h MCAo; 1.8±0.4 at 24 hours (NS between groups). TTC exclusion lesions (% ipsi hemisphere, mean±SE ) were smaller after TH: 15±3 vs 30±4 (p<0.002, t-test). Conclusions: A novel perivascular cooling catheter faithfully simulates endovascular cooling in a rat MCAo model. Filament removal simulates recanalization as would occur during IAT. The combination of recanalization after 4h MCAo and only 2h TH yielded highly significant neuroprotection, with a 50% treatment effect. Future clinical trials may consider combining brief, deep TH with IAT.
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