Cyclin-dependent kinases as a therapeutic target for stroke

Cyclin-dependent kinases (CDKs) are commonly known to regulate cell proliferation. However, previous reports suggest that in cultured postmitotic neurons, activation of CDKs is a signal for death rather than cell division. We determined whether CDK activation occurs in mature adult neurons during focal stroke in vivo and whether this signal was required for neuronal death after reperfusion injury. Cdk4/cyclin D1 levels and phosphorylation of its substrate retinoblastoma protein (pRb) increase after stroke. Deregulated levels of E2F1, a transcription factor regulated by pRb, are also observed. Administration of a CDK inhibitor blocks pRb phosphorylation and the increase in E2F1 levels and dramatically reduces neuronal death by 80%. These results indicate that CDKs are an important therapeutic target for the treatment of reperfusion injury after ischemia.

ZK200775: A phosphonate quinoxalinedione AMPA antagonist for neuroprotection in stroke and trauma

Stroke and head trauma are worldwide public health problems and leading causes of death and disability in humans, yet, no adequate neuroprotective treatment is available for therapy. Glutamate antagonists are considered major drug candidates for neuroprotection in stroke and trauma. However, N-methyl-d-aspartate antagonists failed clinical trials because of unacceptable side effects and short therapeutic time window. α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) antagonists derived from the quinoxalinedione scaffold cannot be used in humans because of their insolubility and resulting renal toxicity. Therefore, achieving water solubility of quinoxalinediones without loss of selectivity and potency profiles becomes a major challenge for medicinal chemistry. One of the major tenets in the chemistry of glutamate antagonists is that the incorporation of phosphonate into the glutamate framework results in preferential N-methyl-d-aspartate antagonism. Therefore, synthesis of phosphonate derivatives of quinoxalinediones was not pursued because of a predicted loss of their selectivity toward AMPA. Here, we report that introduction of a methylphosphonate group into the quinoxalinedione skeleton leaves potency as AMPA antagonists and selectivity for the AMPA receptor unchanged and dramatically improves solubility. One such novel phosphonate quinoxalinedione derivative and competitive AMPA antagonist ZK200775 exhibited a surprisingly long therapeutic time window of >4 h after permanent occlusion of the middle cerebral artery in rats and was devoid of renal toxicity. Furthermore, delayed treatment with ZK200775 commencing 2 h after onset of reperfusion in transient middle cerebral artery occlusion resulted in a dramatic reduction of the infarct size. ZK200775 alleviated also both cortical and hippocampal damage induced by head trauma in the rat. These observations suggest that phosphonate quinoxalinedione-based AMPA antagonists may offer new prospects for treatment of stroke and trauma in humans.

Neurotoxicity of advanced glycation endproducts during focal stroke and neuroprotective effects of aminoguanidine.

Cerebral infarction (stroke) is a potentially disastrous complication of diabetes mellitus, principally because the extent of cortical loss is greater in diabetic patients than in nondiabetic patients. The etiology of this enhanced neurotoxicity is poorly understood. We hypothesized that advanced glycation endproducts (AGEs), which have previously been implicated in the development of other diabetic complications, might contribute to neurotoxicity and brain damage during ischemic stroke. Using a rat model of focal cerebral ischemia, we show that systemically administered AGE-modified bovine serum albumin (AGE-BSA) significantly increased cerebral infarct size. The neurotoxic effects of AGE-BSA administration were dose- and time-related and associated with a paradoxical increase in cerebral blood flow. Aminoguanidine, an inhibitor of AGE cross-linking, attenuated infarct volume in AGE-treated animals. We conclude that AGEs may contribute to the increased severity of stroke associated with diabetes and other conditions characterized by AGE accumulation.

Robotic Outcome Assessment in a Non-Human Primate Model of Middle Cerebral Artery Stroke

Stroke is a prevalent disorder with immense socioeconomic impact. A variety of chronic neurological deficits result from stroke. In particular, sensorimotor deficits are a significant barrier to achieving post-stroke independence. Unfortunately, the majority of pre-clinical studies that show improved outcomes in animal stroke models have failed in clinical trials. Pre-clinical studies using non-human primate (NHP) stroke models prior to initiating human trials are a potential step to improving translation from animal studies to clinical trials. Robotic assessment tools represent a quantitative, reliable, and reproducible means to assess reaching behaviour following stroke in both humans and NHPs. We investigated the use of robotic technology to assess sensorimotor impairments in NHPs following middle cerebral artery occlusion (MCAO). Two cynomolgus macaques underwent transient MCAO for 90 minutes. Approximately 1.5 years following the procedure these NHPs and two non-stroke control monkeys were trained in a reaching task with both arms in the KINARM exoskeleton. This robot permits elbow and shoulder movements in the horizontal plane. The task required NHPs to make reaching movements from a centrally positioned start target to 1 of 8 peripheral targets uniformly distributed around the first target. We analyzed four movement parameters: reaction time, movement time (MT), initial direction error (IDE), and number of speed maxima to characterize sensorimotor deficiencies. We hypothesized reduced performance in these attributes during a neurobehavioural task with the paretic limb of NHPs following MCAO compared to controls. Reaching movements in the non-affected limbs of control and experimental NHPs showed bell-shaped velocity profiles. In contrast, the reaching movements with the affected limbs were highly variable. We found distinctive patterns in MT, IDE, and number of speed peaks between control and experimental monkeys and between limbs of NHPs with MCAO. NHPs with MCAO demonstrated more speed peaks, longer MTs, and greater IDE in their paretic limb compared to controls. These initial results qualitatively match human stroke subjects’ performance, suggesting that robotic neurobehavioural assessment in NHPs with stroke is feasible and could have translational relevance in subsequent human studies. Further studies will be necessary to replicate and expand on these preliminary findings.

Computational models for improved diagnosis and prognosis of stroke using robot-based biomarkers

Stroke is a leading cause of death and permanent disability worldwide, affecting millions of individuals. Traditional clinical scores for assessment of stroke-related impairments are inherently subjective and limited by inter-rater and intra-rater reliability, as well as floor and ceiling effects. In contrast, robotic technologies provide objective, highly repeatable tools for quantification of neurological impairments following stroke. KINARM is an exoskeleton robotic device that provides objective, reliable tools for assessment of sensorimotor, proprioceptive and cognitive brain function by means of a battery of behavioral tasks. As such, KINARM is particularly useful for assessment of neurological impairments following stroke. This thesis introduces a computational framework for assessment of neurological impairments using the data provided by KINARM. This is done by achieving two main objectives. First, to investigate how robotic measurements can be used to estimate current and future abilities to perform daily activities for subjects with stroke. We are able to predict clinical scores related to activities of daily living at present and future time points using a set of robotic biomarkers. The findings of this analysis provide a proof of principle that robotic evaluation can be an effective tool for clinical decision support and target-based rehabilitation therapy. The second main objective of this thesis is to address the emerging problem of long assessment time, which can potentially lead to fatigue when assessing subjects with stroke. To address this issue, we examine two time reduction strategies. The first strategy focuses on task selection, whereby KINARM tasks are arranged in a hierarchical structure so that an earlier task in the assessment procedure can be used to decide whether or not subsequent tasks should be performed. The second strategy focuses on time reduction on the longest two individual KINARM tasks. Both reduction strategies are shown to provide significant time savings, ranging from 30% to 90% using task selection and 50% using individual task reductions, thereby establishing a framework for reduction of assessment time on a broader set of KINARM tasks. All in all, findings of this thesis establish an improved platform for diagnosis and prognosis of stroke using robot-based biomarkers.