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.

Fever and brain injury

Elevation of body temperature occurring as a result of hypothalamic coordination of autonomic, neuroendocrine, and behavioral responses in reaction to physiological injury or invasion is generally known as fever. Traditional thought is that the “febrile response” is beneficial in preventing the proliferation of invading microorganisms, but some caregivers consider fever to be harmful and prescribe antipyretic agents and/or physical cooling methods to suppress fever. In their recent publication, Aiyagari and Diringer summarize the data that exists concerning the efficacy of physical and pharmacological treatments in reducing temperature and improving outcome in a variety of acute neurological disorders including stroke, traumatic brain injury, and cardiac arrest.

Several rationales exist for treating fever, including the relief of discomfort associated with fever, reduction of fever-imposed increase in metabolic demand, reduction in morbidity and mortality, reduction of fever-induced cognitive impairment, and prevention of febrile seizures. Most of these rationales are beneficial in theory, but have not been proven in practice. In the case of morbidity/mortality reduction, treatment with antipyretics has been shown to prolong certain infections; similarly, fever is known to improve survival of patients with community acquired pneumonia, Eschericia coli bacteremia, and Pseudomonas aeruginosa sepsis. Compounding these issues is the fact that traditional methods of lowering temperature in febrile patients are ineffective.

Elevated temperature exacerbates neuronal injury caused by cerebral ischemia or traumatic brain injury (TBI) and, conversely, hypothermia acts as a neuroprotectant in such cases. Well-controlled animal models of global and focal ischemia demonstrate a significantly detrimental effect of hyperthermia on clinical outcome and neuropathological changes. Ginsberg and Busto ( 1998 ) list a number of mechanisms through which hyperthermia worsens outcome in cerebral ischemia: increased neurotransmitter release, increased free radical production, opening of the blood-brain barrier, increased depolarizations within the penumbra, impaired brain metabolism and second messenger inhibition, and cytoskeletal degradation. The authors also note that “the action of otherwise neuroprotective drugs in ischemia may be nullified by mild hyperthermia.” Meticulous brain temperature monitoring and treatment of elevated temperature in patients suffering from neurological insult may, therefore, help prevent secondary injury.

Clinical studies of TBI and survivors of cardiac arrest have demonstrated an independent relationship between fever and poor outcome. Although fever is extremely common in neurological intensive care unit patients, the lack of effective fever treatment options has severely limited the availability of data regarding the benefits of fever reduction in such patients. However, recent advances in surface and intravascular cooling devices have lead to improvements in ability to reduce temperature, especially in patients with neurological injuries. An external cooling device known as the Medivance Arctic Sun temperature management system appears to be quite effective at reducing temperature in febrile patients (75% reduction in fever burden) as compared with more traditional means of fever reduction such as air- or water-circulating cooling blankets. Similarly, a newly-devised catheter-based heat exchange system (Cool Line/Cool Gard) has been tested in patients with subarachnoid hemorrhage (SAH), intracerebral hemorrhage (ICH), cerebral infarction, or TBI, showing a 64% reduction in fever burden as compared to the conventional treatment group (antipyretic, cooling blanket, and ice packs). Unfortunately, no data exists concerning these interventions’ impact on outcome.

As cooling devices and methods are improved and proven to be effective, more data concerning the effect of fever reduction on outcome should be forthcoming. Importantly, as Aiyagari and Diringer point out in the conclusion of their review:

“In the absence of conclusive data, the approach to fever management should be based on the balance between the potential for fever to exacerbate brain insults vs. enhance the ability to fight infections. Fortunately, the risk of ongoing brain injury is usually limited to the early phase in the course of most acute insults while the risk of infection rises as time goes on. Thus it would seem reasonable to aggressively control fever during the first few hours to days following ischemic stroke, intracerebral hemorrhage and head injury. Subsequently, aggressive fever control is less likely to be of help and could be detrimental.”

In cryonics patients, infection exacerbation is less important than protecting the brain from injury and warrants immediate induction of rapid cooling to protect patients from injury due to elevated temperatures. The benefits of treating fever in brain injury also highlights the importance of maintaining normothermia, or even hypothermia, in agonal (hypoxic) patients that present for cryopreservation.

Combination therapy: The patient's view

One consequence of the growing understanding of the biochemical pathways involved in brain injury resulting from cardiac arrest, stroke, and brain trauma is that there is an increasing consensus among researchers that combination therapy is the most logical treatment for the multifactorial injury mechanisms responsible for neuronal death. In this context, combination therapy can mean either combining different forms of treatment, such as hypothermia and a neuroprotective agent, or the combination of multiple neuroprotective agents. But despite encouraging results with combination therapy in animal models, and disappointing outcomes for single neuroprotective agents (such as the recent free radical spin trap agent NXY-059) in human clinical trials, there are no indications that the current trend of investigating just one neuroprotective agent will be reversed soon.

One obstacle for successful combination treatment that is not often addressed is that cardiac arrest, stroke, and brain trauma are acute events that do not allow a vocal pro-active role of the patient at the time that this could benefit him. During the immediate post-insult period when the molecular events leading to neuronal death, and even higher brain death, play out, most patients are not able to communicate their wishes, or are in a coma. As a result, the patient is not present at the time when the most important decisions about his survival as a person are being made.

This predicament is different from patients suffering from serious but chronic diseases such as AIDS and cancer. In his book “Surviving Terminal Cancer: Clinical Trials, Drug Cocktails, and Other Treatments Your Oncologist Won’t Tell You About” psychology professor Ben Williams documents how he improved his odds of surviving a glioblastoma multiforme brain tumor by researching and pursuing his own treatment, which consists of a combination of conventional and “alternative” treatments.

Williams’ successful case of personalized combination therapy does not present strict scientific evidence that his treatment is the cause of his remarkable recovery (so far), but it does highlight the general benefits that may be obtained when patients demand some degree of control over their choice of treatments. Williams stresses that patients such as himself may have much to gain, and not much too lose, from pursuing such an experimental “cocktail” approach. A similar situation applies to patients who are at risk of severe brain injury and cannot afford to wait until the mechanisms and comparative efficacies of each individual component of a neuroprotective cocktail have been thoroughly investigated.

How can such an outcome driven treatment of cerebral ischemia gain acceptance? Since the patient will not “be there” to investigate and demand unorthodox experimental treatments, he can only influence his odds by leaving advance directives to medical care givers and relatives to request that such treatments are given to him. Such measures can only have a chance of succeeding, however, if experimental treatment options are documented for these patients.

In contrast, combinational pharmacotherapy and hypothermia have been core components of human cryopreservation stabilization protocol for many years. To date, researchers involved in cryonics have made record achievements in normothermic cerebral resuscitation and (ultra)profound hypothermic resuscitation. The applications of such research should not be limited to minimizing brain injury in cryonics patients but should be shared with the general public to help build a “supply” side of experimental treatments that can be consulted by medical care givers and relatives of the patient.

Individuals who are signed up for cryonics have a personal interest in stimulating such research, its documentation and dissemination because acute insults such as cardiac arrest, stroke, and brain trauma can produce (higher) brain death before the individual will present for human cryopreservation in the future. Indeed, cryonics may offer the only chance of personal survival for patients who are at risk of major brain damage if they are resuscitated and left to live at room temperature.

Leukocytes and cerebral ischemia

In their paper “The role of leukocytes following cerebral ischemia: pathogenic variable or bystander reaction to emerging infarct?” D.F. Emerich et al. review the literature on the involvement of neutrophils in cerebral ischemia:

“We reasoned that if neutrophils play an important pathogenic (i.e., cause-effect) role in the neuronal damage that follows a stroke, then one should expect to find clear evidence that: (1) neutrophils invade the ischemic area prior to terminal stage infarction, (2) greater numbers of early appearing neutrophils are accompanied by evidence of greater neuronal loss, and (3) dose-related inhibition of neutrophil trafficking or activity produces a corresponding decrease in the degree of brain damage following ischemia.”

The authors did not find much evidence for any of the above and speculate that neutrophil recruitment may not be a cause of injury, but rather a response to postischemic necrosis.

Knowledge of the causal and temporal aspects of cerebral ischemia is important to select the right agents to minimize brain injury of cryonics patients. Neuroprotective agents that confer benefits to cardiac arrest and stroke victims may not necessarily offer additional protection during stabilization of cryonics patients if the targets of these interventions are non-causal and/or delayed events in the ischemic cascade.