tat-nr2b9c and Infarction--Middle-Cerebral-Artery

Excitotoxicity mediated by the N-methyl-D-aspartate receptor (NMDAR) is believed to be a primary mechanism of neuronal injury following stroke. Thus, many drugs and therapeutic peptides were developed to inhibit either the NMDAR at the cell surface or its downstream intracellular death-signaling cascades. Nevertheless, the majority of focal ischemia studies concerning NMDAR antagonism were performed using the intraluminal suture-induced middle cerebral arterial occlusion (MCAO) model, which produces a large cortical and subcortical infarct leading to hypothalamic damage and fever in experimental animals. Here, we investigated whether NMDAR antagonism by drugs and therapeutic peptides was neuroprotective in a mouse model of distal MCAO (dMCAO), which produces a small cortical infarct sparing the hypothalamus and other subcortical structures. For establishment of this model, mice were subjected to dMCAO under normothermic conditions or body-temperature manipulations, and in the former case, their brains were collected at 3-72 h post-ischemia to follow the infarct development. These mice developed cortical infarction 6 h post-ischemia, which matured by 24-48 h post-ischemia. Consistent with the hypothesis that the delayed infarction in this model can be alleviated by neuroprotective interventions, hypothermia strongly protected the mouse brain against cerebral infarction in this model. To evaluate the therapeutic efficacy of NMDAR antagonism in this model, we treated the mice with MK801, Tat-NR2B9c, and L-JNKI-1 at doses that were neuroprotective in the MCAO model, and 30 min later, they were subjected to 120 min of dMCAO either in the awake state or under anesthesia with normothermic controls. Nevertheless, NMDAR antagonism, despite exerting pharmacological effects on mouse behavior, repeatedly failed to show neuroprotection against cerebral infarction in this model. The lack of efficacy of these treatments is reminiscent of the recurrent failure of NMDAR antagonism in clinical trials. While our data do not exclude the possibility that these treatments could be effective at a different dose or treatment regimen, they emphasize the need to test drug efficacy in different stroke models before optimal doses and treatment regimens can be selected for clinical trials.

Topics: Animals; Cerebral Infarction; Disease Models, Animal; Dizocilpine Maleate; Hypothermia, Induced; Infarction, Middle Cerebral Artery; Male; Mice; Neuroprotective Agents; Peptides; Receptors, N-Methyl-D-Aspartate; Treatment Outcome

Following positive results with the poly-arginine peptide R18 when administered intravenously 30 or 60min after permanent and/or transient middle cerebral artery occlusion (MCAO; 90min) in the rat, we examined the effectiveness of the peptide when administered 2h after MCAO. R18 was administered intravenously (1000nmol/kg via jugular vein) after permanent MCAO or a transient 3-h MCAO or when administered intra-arterially (100nmol/kg via internal carotid artery) immediately after reperfusion following a transient 2-h MCAO. In the transient MCAO studies, the neuroprotective NA-1 peptide was used as a positive control. Infarct volume, cerebral edema and functional outcomes were measured 24h after MCAO. Following permanent or transient MCAO, neither R18 nor NA-1 significantly reduced infarct volume. However, following permanent MCAO, R18 appeared to reduce cerebral edema (p=0.006), whereas following a transient 3-h MCAO, R18 improved the time to remove adhesive tape (p=0.04) without significantly affecting cerebral edema. There was also a trend (p=0.07) towards improved rota-rod performance with R18 in both permanent and transient 3-h MCAO. Following a transient 2-h MCAO, R18 had no significant effects on cerebral edema or neurological score but did lessen the extent of weight loss. Overall, while R18 had no effect on infarct volume, the peptide reduced cerebral edema after permanent MCAO, and improved some functional outcomes after transient MCAO.

Topics: Animals; Brain Edema; Brain Ischemia; Carotid Artery Injuries; Carotid Artery, Internal; Cerebral Arteries; Cerebrovascular Disorders; Disease Models, Animal; Infarction, Middle Cerebral Artery; Intracellular Signaling Peptides and Proteins; Ischemic Attack, Transient; Male; Neuroprotective Agents; Peptides; Rats; Reperfusion; Stroke

We examined the efficacy of R18 in a transient MCAO model and compared its effectiveness to the well-characterized neuroprotective NA-1 peptide. R18 and NA-1 peptides were administered intravenously (30, 100, 300, 1000nmol/kg), 60min after the onset of 90min of MCAO. Infarct volume, cerebral swelling and functional outcomes (neurological score, adhesive tape and rota-rod) were measured 24h after MCAO. R18 reduced total infarct volume by 35.1% (p=0.008), 24.8% (p=0.059), 12.2% and 9.6% for the respective 1000 to 30nmol/kg doses, while the corresponding doses of NA-1 reduced lesion volume by 26.1% (p=0.047), 16.6%, 16.5% and 7%, respectively. R18 also reduced hemisphere swelling by between 46.1% (1000 and 300nmol/kg; p=0.009) and 24.4% (100nmol/kg; p=0.066), while NA-1 reduced swelling by 25.7% (1000nmol/kg; p=0.054). In addition, several R18 and NA-1 treatment groups displayed a significant improvement in at least one parameter of the adhesive tape test. These results confirm the neuroprotective properties of R18, and suggest that the peptide is a more effective neuroprotective agent than NA-1. This provides strong justification for the continuing development of R18 as a neuroprotective treatment for stroke.

Topics: Animals; Brain Edema; Brain Infarction; Disease Models, Animal; Dose-Response Relationship, Drug; Infarction, Middle Cerebral Artery; Movement; Nervous System Diseases; Neuroprotective Agents; Peptides; Psychomotor Performance; Rats; Statistics, Nonparametric; Treatment Outcome

Tat-NR2B9c, a clinical-stage stroke neuroprotectant validated in rats and primates, was recently deemed ineffective in mice. To evaluate this discrepancy, we conducted studies in mice subjected to temporary middle cerebral artery occlusion (tMCAO) for either 30 or 60 min according to the established principles for dose-translation between species. Tat-NR2B9c treatment reduced infarct volume by by 24.5% (p = 0.49) and 26.0% (p = 0.03) for 30 and 60 min tMCAO, respectively, at the rat-equivalent dose of 10 nMole/g, but not at the previously reported 3 nMole/g in mice. Dose translation is thus critical when preclinical experiments are conducted in new species.

Topics: Animals; Brain; Disease Models, Animal; Dose-Response Relationship, Drug; Infarction, Middle Cerebral Artery; Male; Mice; Mice, Inbred C57BL; Neuroprotective Agents; Peptides; Rats; Species Specificity

All attempts at treating strokes by pharmacologically reducing the human brain's vulnerability to ischaemia have failed, leaving stroke as a leading cause of death, disability and massive socioeconomic loss worldwide. Over decades, research has failed to translate over 1,000 experimental treatments from discovery in cells and rodents to use in humans, a scientific crisis that gave rise to the prevailing belief that pharmacological neuroprotection is not feasible or practicable in higher-order brains. To provide a strategy for advancing stroke therapy, we used higher-order gyrencephalic non-human primates, which bear genetic, anatomical and behavioural similarities to humans and tested neuroprotection by PSD-95 inhibitors--promising compounds that uncouple postsynaptic density protein PSD-95 from neurotoxic signalling pathways. Here we show that stroke damage can be prevented in non-human primates in which a PSD-95 inhibitor is administered after stroke onset in clinically relevant situations. This treatment reduced infarct volumes as gauged by magnetic resonance imaging and histology, preserved the capacity of ischaemic cells to maintain gene transcription in genome-wide screens of ischaemic brain tissue, and significantly preserved neurological function in neurobehavioural assays. The degree of tissue neuroprotection by magnetic resonance imaging corresponded strongly to the preservation of neurological function, supporting the intuitive but unproven dictum that integrity of brain tissue can reflect functional outcome. Our findings establish that tissue neuroprotection and improved functional outcome after stroke is unequivocally achievable in gyrencephalic non-human primates treated with PSD-95 inhibitors. Efforts must ensue to translate these findings to humans.

Topics: Animals; Brain; Disks Large Homolog 4 Protein; Humans; Infarction, Middle Cerebral Artery; Intracellular Signaling Peptides and Proteins; Macaca fascicularis; Magnetic Resonance Imaging; Male; Membrane Proteins; Peptides; Stroke; Time Factors; Treatment Outcome