Tag: Neuroprotectant

Neuroprotection for ischemic stroke

The journal Neuropharmacology recently published a new review of the current state of the art in neuroprotection for ischemic stroke. A strict definition of a neuroprotectant excludes agents that have as their goal circulatory patency or the reversal of vascular occlusion, such as thrombolytics and anticoagulants. As a consequence, the only medication that is approved for (ischemic) stroke patients, tPA, is not a neuroprotectant. Despite the explosion of interest and research in the field (as documented in Ginsberg’s review), no single neuroprotective agent has successfully survived human clinical trials. The author discusses a number of reasons why encouraging results fail to translate into human success and stresses the fact that most agents in clinical trials are administered too late to confer positive benefits, and even states that “there is practically no evidence that neuroprotection for acute ischemic stroke is possible with any agent beyond ~6h.” It is no surprise, then, that the author does not report many promising neuroprotective strategies except for therapeutic hypothermia, high-dose human albumin therapy, and hyperacute magnesium therapy.

What does this mean for cryonics? As discussed in this review about medications in human cryopreservation stabilization, neuroprotection in cryonics has never been approached as a quest to find one single “magic bullet” to protect the brain after cardiac arrest. Cryonics stabilization medications protocol consists of a number of agents that intervene at different points in the ischemic cascade, reverse and inhibit blood clotting, and improve circulation. If rapid stabilization is possible, the time-window for treatment in cryonics is usually excellent in comparison to (focal) ischemic stroke where treatment within 1-2 hours is considered “hyperacute.” But cardiac arrest after an (often) long terminal and agonal period is not equivalent to (focal) ischemic stroke, and evidence that the medications that are given to cryonics patients are of great benefit is confined to a series of (non-published) experiments on (young) healthy animals in cryonics-associated laboratories.

When the author discusses future directions to find successful neuroprotective agents, he highlights the challenge of finding funding for neuroprotective trials that include metabolic treatment and combinations of (non-proprietary) drugs. In light of the predictable failure of mono-agents that the author reports, the discussion of the potential of combination treatment is remarkably brief and confined to the point that potential neuroprotectants need to be validated in combination with thrombolytic treatment. There is now an accumulating number of research papers on combination treatment in animal models that would warrant a more systematic analysis than the obligatory acknowledgement that combination therapy might produce better neuroprotection. Perhaps the most novel part of this new review of neuroprotective agents is the discussion of the author’s own research into high-dose human albumin therapy and the brief mention of a new paper (2007) that discusses the prospects of neuroprotective strategies that “are based on the principle that drugs should be activated by the pathological state that they are intended to inhibit.”

Incomplete assessment of experimental cytoprotectants in rodent ischemia studies

In an effort to determine why so many cytoprotective treatments for stroke that are shown to be promising in laboratory animal experiments ultimately fail in human clinical trials, DeBow et al. performed an analysis of cytoprotection studies published in several leading journals. While noting that limitations in preclinical assessments also contribute to the premature advancement of some therapies to the clinic, their primary goal was to pinpoint deficiencies in experimental methodology of rodent ischemia studies that might lead to this inability to translate positive results from the bench to the bedside.

Because the stroke therapy academic industry roundtable (STAIR) issued a report in 1999 recommending certain improvements in stroke research, the authors decided to include literature beginning in 2000 up to the time of their analysis in 2002, to be compared with the representative literature in 1990. They identified all of the rodent ischemia articles published in the Journal of Neuroscience, the Journal of Cerebral Blood Flow and Metabolism, Experimental Neurology, and Stroke, including only studies that conformed to the following criteria: (1) used adult to aged rodents to assess global or focal ischemic insults, (2) tested a putative “cytoprotective” therapy, defined as one in which a therapy was administered or a manipulation was made (e.g., knockout mouse) and (3) the effects of that therapy were assessed on histological and/or behavioral outcome.

Following those criteria, the authors identified 19 and 20 global ischemia experiments for 1990 and 2000-2002, respectively. They identified 6 and 118 focal ischemia experiments for 1990 and 2000-2002, respectively. They further categorized these studies by rodent species, sex, and age. Methods used to measure intra- and postsurgery temperature were categorized according to location (rectal, temporalis muscle/tympanic, or brain temperature) and frequency (continual, frequent, infrequent, or none) of measurement. Survival time, measured in terms of hours or days following the start of ischemia, was categorized, as was behavioral evaluation (absent, neurologic deficit scale (NDS), or additional testing with or without NDS).

Almost all studies reviewed reported positive results. The vast majority of studies used male rodents, ignoring possible gender differences, and only two recent studies used old animals (> one year old), indicating that most models do not accurately reflect the typical human clinical situation. Additionally, the authors reported that a full eighty percent of recent (2000-2002) global ischemia experiments used survival times of ≤ seven days, while sixty-six percent of recent focal ischemia studies used survival times of ≤ 48 hours. Only 8.5% of focal ischemia studies examined histological outcome after seven days, essentially the same ratio observed in 1990. Furthermore, very few of the global ischemia studies, recent (2 in 20) or old (1 in 19), assessed behavior after ischemia. The authors comment:

In the recent focal ischemia studies, 55.1% did not assess functional outcome, 33.9% used NDS alone, and 11.0% used additional testing (e.g., skilled reaching) with or without a NDS. None of the 1990 studies used behavioral assessment as an endpoint.

Concerning temperature measurement, the authors found that the majority of global and focal ischemia studies used either rectal or core temperature measurements during ischemia without any other means of predicting brain temperature. Although they noted that some studies measured temporalis muscle (skull) temperature, very few studies directly measured brain temperature. Only 15% of recent global ischemia studies and 2.5% of recent focal ischemia studies utilized telemetry probes; they were not utilized at all in the 1990 papers surveyed.

Similarly, wide variations in postsurgical temperature measurement were reported. Three of the 19 global ischemia studies surveyed from 1990 reported rectal temperatures for up to 2, 6, or 24 hours. Three of the 20 global ischemia studies surveyed from 2000-2002 measured temperature continually with telemetry probes for at least 24 hours, while three other studies from the same time period sampled rectal temperature up to one or two hours following ischemia. In general, postsurgical temperature measurement was largely not performed. The authors report that “The percentage of cytoprotection studies in focal ischemia that measured temperature following surgical anesthesia, even if once, was only 0% and 33.0% for 1990 and 2000-2002, respectively.” Several other studies of both types of ischemia and from both time periods reported placement of animals in temperature-controlled rooms without measuring the animals’ temperatures.

It is not surprising, after reviewing these results, to learn that many of the “cytoprotectants” found to be beneficial in rodent ischemia studies go on to fail human clinical trials. This survey of rodent ischemia studies clearly demonstrates that most current experimental studies do not accurately represent clinical conditions of ischemia (e.g., aged animals) and have serious methodological limitations and flaws that will continue to contribute to clinical failures.

Especially concerning is the fact that most of these studies used exceedingly limited survival times, which are not sufficient to allow injury to mature fully. Exacerbating this issue is the fact that few studies assess behavior after treatment. Such a deficiency may lead an investigator to overestimate the benefit of their treatment since not all reductions in cell death will translate into improved functional outcome. Those studies that did include behavioral assessments typically used only a NDS soon after injury, resulting in a host of other difficulties in determination of actual cytoprotectant effect.

The importance of temperature in cerebral ischemic injury is well documented. Most studies assessed temperature during ischemia, but few measured postsurgical temperature, which is also known to substantially modify ischemic brain damage. The authors state:

Given these findings and the possibility of drug interactions, it is remarkable that most studies did not assess postsurgical temperature at all, including those using drugs known to affect temperature. Furthermore, of those that did, many only took rectal probe measurements for a short period following ischemia, or sampled temperature too infrequently (e.g., one sample at 24 hr after middle cerebral artery occlusion) or not long enough following drug administration (e.g., 15 min) to rule out temperature confounds.

While success rates of cytoprotectants to treat stroke depend not only on experimental design flaws but also limitations and flaws in clinical studies (let alone the relevance of rodent studies to humans), this review does much to show that many investigators have not taken it upon themselves to improve their study designs to avoid confounds or to better represent clinical conditions. Without these improvements, treatments based on such studies will continue to fail.

Because future changes or improvements to cryonics stabilization protocol may include results obtained from rodent ischemia research, general improvements in experimental design will benefit cryonics patients. More specific benefits may be achieved by using models that better reflect a typical cryonics case (e.g., warm ischemia followed by a period of low-flow reperfusion and concurrent temperature reduction). Of course, impeccable temperature monitoring is absolutely critical to such cryonics-specific models, allowing the researcher to control for temperature-related post-surgical pathologies and to better determine the effect of cytoprotective drugs vs. induction of hypothermia.

Systemic administration of L-Kynurenine

L-Kynurenine (L-KYN) is one of the neuroprotective agents used in cryonics stabilization protocol to limit injury to the brain after cardiac arrest. Administration of L-KYN was perceived to be essential to resuscitate dogs from extended periods (up to 17 minutes) of normothermic ischemia during the Critical Care Research (CCR) cerebral resuscitation experiments in the late 1990s. In cryonics L-KYN has been combined with another neuroprotective agent, niacinamide, to make the compound NiKy.

L-KYN is the precursor of kynurenic acid, the only known endogenous antagonist of the excitatory amino acid receptors. Unlike kynurenic acid, L-KYN can cross the blood brain barrier. Because in cryonics neuroprotective agents are administered to the systemic circulation, the effects of such molecules on blood pressure and cerebral blood flow must be weighed against the benefits as a neuroprotectant.

Katalin Sas et al. investigated the effect of systemic administration of L-Kynurenine on corticocerebral blood flow under normal and ischemic (unilateral carotid artery occlusion) conditions in conscious rabbits. Corticocerebral blood flow (cCBF) was measured using the hydrogen clearance technique (i.e., hydrogen polarography). The investigators observed a significant increase in cCBF for both normal and ischemic animals. In the ischemic animals systemic administration of L-KYN resulted in cCBF that approached or even exceeded the base values measured in the normal animals. Administration of L-KYN did not alter arterial blood pressure or heart rate and its effects were of long duration, peaking between 60 and 240 minutes after administration.

The experiments cannot answer the question of whether L-KYN itself or one of its derivatives (such as kynurenic acid) increases cCBF. Other NMDA receptor antagonists have been found to increase cCBF as well. The authors investigated the possibility that the increase of cCBF might be caused by activation of the ascending cholinergic pathways as a response to NMDA receptor antagonism and found that pre-treatment with atropine prevented the increase of cCBF by L-KYN. The authors also investigated the effect of pre-treatment with the non-specific nitric oxide synthase (NOS) inhibitor L-NAME and found that the increase of cCBF was blocked by this agent as well. This suggests that the L-KYN induced increase in cCBF may be mediated by NMDA antagonist induced NO production. A direct effect of L-KYN on cerebral vessels is doubtful because other studies using either glutumate, NMDA, or agonists and antagonists of the former, failed to affect the tone of isolated cerebral arteries.

If systemic administration of L-KYN enhances cCBF in humans as well, L-KYN might be an attractive agent to treat stroke and cardiac arrest due to its multimodal properties. The beneficial properties of L-KYN on cCBF, instead of (or in addition to) its properties as a neuroprotectant may explain its importance in the CCR cerebral resuscitation experiments. Unlike some neuroprotective agents used in cryonics, such as Propofol and Tempol, L-KYN does not appear to have adverse hemodynamic effects and even improves cerebral blood flow. Although the efficacy of kynurenine as a neuroprotectant in cryonics remains uncertain and investigations into the biochemical and temporal aspects of its metabolism (and the effects of rapid induction of hypothermia on this) are warranted, the drug cannot be ruled out because of adverse effects on blood pressure or cerebral blood flow.