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.