Introduction
One of the primary reasons scientists perform post- stroke recovery research on rodents is to determine the usefulness or efficacy of certain treatments or therapies.
The foundation for the validity of animal research is the extended common history that other animals share with our species and the relatively conservative process by which evolutionary change takes place (Chapter 7).
Thus there should be constancy in cerebral organiza- tion and function, both between and within mamma- lian species (Chapter 1). That said, the failure of clinical trials for certain treatments that were effective in ani- mal models has raised an opposing view, expressed in its most extreme form as “animal research tells us nothing about whether a treatment will be effective in people.”Whereas some postulated reasons for the fail- ure of the clinical trials have been summarized else- where [1], here we offer a series of considerations that should be well contemplated when adopting an animal model to investigate whether a certain treatment or therapy will be effective and potentially translate to people that have sustained a stroke. The considerations fall into four broad categories: intervention issues, organismal factors, modeling stroke, and measure- ment. We also review four categories of treatments and therapies that are utilized in animal models to facilitate recovery: enrichment/experience, pharmaco- therapy, cell-based therapies, and electrical stimulation.
Considerations (Table 4.1)
Timing and intensity of intervention
When a cerebral attack or stroke occurs there is a non- trivial interruption of the blood supply to one of the cerebral arteries. This sets off a domino-like cascade of neurodegenerative changes that develop over several timescales (Figure 4.1). The drop in oxygen
concentration causes changes in the ionic balance of the affected regions over thefirst seconds to minutes, including changes in pH and properties of the cell membrane. These ionic changes result in a variety of pathological events, including the release of excessive amounts of glutamate and the prolonged opening of calcium channels. The open calcium channels allow high levels of calcium to enter the cell, which in turn leads to a number of toxic effects that result in cell death. The development of considerable edema (swel- ling) is a major complication over thefirst day follow- ing stroke. The swelling itself can cause neuronal injury, dysfunction, and death. These post-stroke changes in neuronal functioning lead to a drop in metabolic rate and/or glucose utilization in the injured hemisphere that may persist for days. Moreover, areas that receive synaptic input from the primarily dam- aged area suffer a sudden withdrawal of excitation or inhibition. Such sudden changes in input lead to a greater loss of function and secondary cell death that may be even more pronounced than the cell death resulting directly from the injury itself. Following cell death, inflammatory processes begin almost immedi- ately. Microglia invade the damaged region via the vascular system and clear away degenerative debris, a process that may take months to complete. Astrocytes adjacent to the lesioned area enlarge and extend fibrous processes that serve to isolate the surfaces of the injury from the surrounding tissue. Stem cells may also be stimulated to increase division in the subven- tricular zone and to migrate to the injury, although we do not yet know what function these cells might have.
Thus, when considering a treatment, the timing of the intervention in relation to the post-stroke neurodege- nerative cascade must be evaluated.
Treatments for cerebral injury can be targeted at different aspects of the neurodegenerative cascade.
Agents that are designed to protect neurons from the
Brain Repair After Stroke, ed. S. C. Cramer and R. J. Nudo. Published by Cambridge University Press.
© Cambridge University Press 2010.
cascade of toxic events that follow an ischemic episode are called neuroprotectants. For example, drugs can be used to block calcium channels or prevent ionic imbal- ance. There are also new classes of drugs targeting novel channels. One example is the transient receptor potential (trp) channels that mediate the response of a cell to extracellular environmental changes by increas- ing or decreasing the selective permeability to partic- ular ions. Lowering of temperature and thus slowing metabolism is another promising immediate post- stroke treatment [2]. Obviously thefield of neuropro- tection is critically important and should receive more inquiry.
Treatments that make use of plasticity, the endog- enous ability to change the structure and function of the brain, are normally initiated some time after the ischemic insult. It is important to consider that if initiated in the immediate post-injury period, some types of activities might actually make cell death worse or interfere with beneficial plastic changes.
During the course of studies investigating the promo- tion of functional recovery after injury, Schallert and his colleagues accidentally found that initiating intense therapy soon after a stroke worsened the damage [3]
(see Chapter 8). In these studies, rats werefitted with a restraint harness that prevented them from using the forelimb ipsilateral to a sensorimotor cortex injury, thus forcing use of the impaired limb. Unfortunately, the rats that had continual forced use of the affected limb showed significantly enlarged lesion cavity and
worse functional outcome. Although few human stroke treatments would be this intense, the Schallert studies focus attention on the question of when therapy should commence and how intense it should be.
Related to the timing of the treatment or therapy is the intensity or amount of intervention. Our field is not sufficiently advanced to enable us to know a priori the amount of drug or intensity of therapy that will have the optimal effect. Similar to deriving a dose– response curve, researchers need to assess a series of different intensities of therapies for particular patient groups. Moreover, individual differences in responses should also be considered. There is growing evidence that patients who are placed in a dedicated stroke unit, rather than being treated on an outpatient basis, are likely to show a better outcome because they receive more intensive treatment from a variety of healthcare professionals.
Organismal factors
Age
Strokes can occur at any age with a relatively high incidence of cerebral attack in the young, usually at birth. However, most people that sustain and survive a stroke are aged. It has long been known that, on average, children seem to have a better outcome after injury than adults. Kolb has examined the behavior of adult rats that received focal injuries to the medial prefrontal, motor, temporal, posterior parietal, or
Figure 4.1 Time course of post-stroke events in the neurodegenerative cascade.
posterior cingulate cortex on postnatal days 1, 4, 7, 10, or 90 (i.e., adult) [4]. The overall result was that regardless of the location of injury, the functional out- come was always best after injury sustained during the second week of life, which in the rat is a time of intense cerebral synaptogenesis and glial formation. The take- home message is that it is the stage of neural develop- ment, and not age per se, that is the important variable in recovery.
Injured young brains have been shown to compen- sate for lost tissue in three ways: (1) reorganization of existing neuronal networks; (2) development of novel networks; and (3) regeneration of the lost tissue (which is dealt with later in the chapter). Existing normal circuits are found to reorganize following uni- lateral damage to motor systems. When damage occurs to the cortex that normally gives rise to the corticobulbar and corticospinal pathways, the intact pathway on the opposite side sprouts both an enlarged ipsilateral corticospinal pathway as well as new con- nections to subcortical motor regions of the damaged hemisphere [5]. Similar findings are seen in sensory systems as novel pathways develop after damage [6].
Neuronal network remodeling in the form of changes to dendritic morphology have also been observed after early injuries. The overall result of these studies is that when functional outcome is positive, there is an increase in dendritic arborization and spine density in pyramidal neurons in the remaining cortex. When functional outcome is poor, however, there is an atro- phy of dendritic arbor and spine density.
It is generally assumed that as animals age they become less plastic. Teuber reported that brain- injured soldiers also showed a benefit of younger age:
18-year-olds fared better than 25-year-olds, who in turn fared better than older soldiers [7]. Although it has been shown that older animals demonstrate less synaptic potentiation in response to high-frequency stimulation [8], even senescent animals can show con- siderable cortical plasticity [9]. Systematic studies of cerebral plasticity and behavior throughout the life span in both normal and brain-injured animals are still required. Moreover, the health status and life- history of experience rather than the age of the animal may be the predominant factor.
Comorbid conditions
Most people that survive a stroke often have a variety of other pre-existing medical conditions such as dia- betes, arthritis, atherosclerosis, and hypertension that
need to be modeled by stroke researchers. For instance, a clinically relevant model of human stroke is the stroke-prone spontaneously hypertensive rat.
This strain of rat has been shown to have a genetic predisposition to cerebral ischemia and exhibits hypertension and an increased sensitivity to experi- mentally induced stroke. Following stroke, these rats exhibit greater impaired functional recovery com- pared to a normotensive strain from which the hyper- tensive strain was derived [10]. The usage of animal models that replicate at least some of the more preva- lent comorbid conditions in people that have had strokes will need to be more fully embraced by researchers.
Most stroke survivors take numerous medications for a variety of ailments and conditions. For instance, because clinical depression is found in the majority of stroke survivors (see Chapter 14), the prescription of specific serotonin reuptake inhibitors (SSRIs) has been almost universal where available. SSRIs have been shown to alter brain activity and modulate motor performance in stroke patients in a use-dependent fashion. Moreover, several antidepressants, including fluoxetine, increase growth factors and other proteins associated with plasticity, such as brain-derived neuro- trophic factor (BDNF). In one study, however, the addition offluoxetine treatment to rehabilitation ther- apy did not alter the degree or rate of recovery of function compared to non-treated animals [11]. In another study,fluoxetine did not affect sensorimotor or water-maze performance in aged rats after exper- imental stroke [12]. The ability offluoxetine to alter brain activity and increase growth factors does not appear to be an effective pharmacological adjunct to rehabilitative therapy after ischemia in rats.
Seizures are observed following strokes in people and are also commonly observed following the crea- tion of a stroke in animal models. Many investigators ignore the seizures and treat them as a nuisance.
However, the duration and severity of seizures can dramatically affect stroke size and behavioral outcome, with long seizures associated with a negative outcome.
Seizures can dramatically change the balance between excitation and inhibition, resulting in reorganized movement representations [13] and sensory function [14]. Moreover, seizures are known to reorganize cir- cuits through Hebbian-like algorithms due to the coin- cident firing of pre- and postsynaptic neurons that occurs during an ictal event. Thus, seizures may“use up”plastic capacity, leaving the brain less responsive
to treatments and therapies. Alternatively, brief and mild seizures are known to stimulate the production and release of growth factors, which in turn could support vulnerable neurons, prevent cell death and assist in the formation of new circuits. A common view in the medical community seems to be that all seizures are bad and should be treated with anticon- vulsants. This leads to two problems: (1) not all seiz- ures may be associated with negative outcomes and may in certain circumstances be beneficial or at least innocuous, and (2) anticonvulsant medications them- selves are known to be associated with developmental delays and poorer functional recovery [15]. The take- home message on seizures is that they should not be ignored in experimental settings because they represent an important variable that needs to be understood.
Sex and hormonal status
Males and females have been shown to have different responses to brain injury and post-injury treatments.
For example, the effect of frontal injury in both humans and rats is more severe in females than in males [4]. When rats with similar frontal injuries early in development were placed in complex environ- ments, there was a greater benefit of the treatment on cognitive functions for male rats than for females, whereas the opposite pattern of results was found for motor functions [16]. Moreover, the cyclic nature of hormone levels and hormonal status (e.g. menopause) can also influence plasticity and, thus, recovery pro- cesses [17]. How the existing medical condition, med- ications and brain injury interact will likely be an extremely complex puzzle to untangle.
Modeling stroke
Occlusion of the middle cerebral artery (MCA), which is responsible for the majority of thrombotic strokes in humans, is the most widely employed model of stroke using rodents. This leads to a problem for those of us interested in functional recovery. The problem is that the infarct volume following MCA occlusion in rodents is proportionately very much larger than that found in humans, always involves both gray and white matter, and is devastating from a behavioral perspec- tive. Thus, while the MCA occlusion has face validity, it is not a good choice for those interested in examin- ing behavioral recovery. There are other methods to induce focal ischemic infarcts that can be fairly pre- cisely localized to specific brain regions that leave
substantial tissue intact that will support functional recovery. These methods can also be used to lesion either gray or white matter (or both) and to target specific locations. The location of the stroke and type of impairment is also important, because this deter- mines whether the infarct gives rise to sensory, motor, or cognitive impairment.
People who survive strokes vary in the degree of impairment from mild to catastrophic (see Figure 4.2).
Those with mild impairments often make a full recov- ery to the point that they do not manifest obvious impairment of function. At the opposite end of the spectrum there are individuals who survive a stroke that will be maintained on artificial life support for the remainder of their life. In the laboratory, treatments are usually tested for efficacy in animals with moderate impairments, while severely and mildly impaired animals are removed from the study for ethical and practical reasons. Researchers should consider exam- ining the amount of impairment on an individual animal basis to determine the relationship between the degree of impairment and the efficacy of the therapy. This is an important consideration from the perspective of industry, as they are interested in identi- fying individuals that will reap the most benefit from their treatment.
Measurement issues
Researchers routinely report quantitative end-point measures of the behaviors of their subjects. End- point measures, such as latency to initiate a movement or duration to complete a task, or percent success at
Mild
Moderate
Severe
Time
Performance (%)
100
80
60
40
20
0
Figure 4.2 Amount of available recovery post-stroke. Animals with mild strokes recover fully but can only show small improvements.
Animals with moderate strokes can show substantial recovery.
Animals with severe strokes often do not recover and show progressive deterioration.
retrieving a pellet, are three common examples. While end-point measures are important, they capture only a sliver of the whole behavior of the animal. Taking a more comprehensive approach to behavioral measure- ment can lead to important insights. For instance, Whishaw and colleagues developed a 10-point kine- matic analysis of how rats use their forelimb when retrieving a food pellet [18]. Brain-injured rats often perform differently from controls on the task even though their end-point measures may be equivalent.
Thus, a wealth of important data that are available to
the researcher should be utilized. Unfortunately, the measurement sophistication and detail in the clinic are often quite crude and are lagging behind those employed by animal researchers in the lab.
Each mammalian species has a behavioral reper- toire available to it. The repertoire represents the whole range of behaviors supported by their brains and bodies. Rats in particular are highly skilled at reaching and performingfine and dexterous manipu- lations with their forelimbs [19]; thus, they provide wonderful models for behavioral study. However,
Table 4.1 Several considerations when modeling post-stroke treatment or therapy in animals.
Consideration category
Subcategories Specifics
Intervention
Timing of intervention
How long after stroke
Intensity of intervention
How much of the day and for how long
Persistence of treatment effect
Duration of benefit
Organismal factors
Age at time of stroke
Newborn, young, adult, aged
Comorbid conditions
Other medical conditions (atherosclerosis, coronary disease, diabetes, hypertension), medications, seizures
Sex Male, female
Hormonal status Juvenile, adult, post-menopausal Modeling strokes
Type of stroke Global, focal (thrombotic, hemorrhagic) Location of stroke Gray matter, white matter, mixed Kind of
impairment
Motor, sensory, cognitive, mixed
Degree of impairment
Severe, moderate, mild
Stroke induction methodology
Arterial occlusion, focal (pial strip, electrocautery, photothrombotic)
Measurement
Species Size of brain (proportion of gray and white matter), available behavioral repertoire, practical (cost) and ethical (companion animals) concerns
Measurement End points of success, behavioral strategies (recovery or compensation)
there are important structural differences in the shoul- der and wrist anatomy between rats and primates (including humans) and the differences must be accounted for when drawing parallels to humans.
Simply put, species differences in sensory, cognitive, and motor function (e.g. bees have a visual range that includes the ultraviolet and dogs lack color vision) must be considered when examining and interpreting their behaviors.
Enrichment/experience
Studies of laboratory animals have consistently shown that the single most successful treatment strategy for optimizing functional recovery from a variety of forms of experimental brain injuries is placing animals in complex, stimulating environments [4,20]. Although the mechanisms responsible for the beneficial effects of complex housing are not fully known, it has been hypothesized that the treatment may increase the syn- thesis of neurotrophic factors, which in turn facilitate synaptic plasticity. Motor training has been shown to upregulate trophic factors such as BDNF and basic fibroblast growth factor (bFGF or FGF-2) [21], so we might anticipate that rehabilitative motor training after cerebral injury would also be beneficial. There is some evidence of benefits from repetitive motor training [22], and this type of training is often used by physiotherapists. Such treatments have not always been found to be beneficial, however, and the differences may be related to the details of the training.
Pharmacotherapy
Drug therapies can provide a relatively easy and cost- effective means of facilitating plastic changes in the injured brain that would support functional improve- ment. Psychomotor stimulants such as amphetamine or nicotine are known to stimulate changes in cortical and subcortical circuits in the normal brain. It is thus reasonable to suppose that these agents could stimu- late plastic changes in the injured brain to facilitate recovery. Nicotine appears to facilitate recovery from strokes in motor regions and does so by supporting synaptic changes in spared motor regions [23]. These changes are correlated with both qualitative and quan- titative changes in behavior. Amphetamine has also been shown to be beneficial in rats, but has had mixed clinical success. Kolb and colleagues have compared the effects of amphetamine on recovery from focal versus more extensive strokes [24]. Amphetamine
was effective in producing both synaptic change and behavioral improvement after focal cortical injuries, but showed little benefit after large middle cerebral occlusions. In contrast, nicotine still produced some benefit after larger strokes. One important difference between the drugs is that nicotine has more wide- spread effects on cortical circuitry than does amphet- amine [25], a difference that may account for the added benefits of nicotine after cerebral injury.
However, it should be recognized that the effect of nicotine was studied by giving rats nicotine alone and not in conjunction with smoke and other contam- inants related to taking nicotine by smoking tobacco.
It seems likely that post-injury smoking would not be the ideal treatment, especially after stroke. Moreover, the nicotine was delivered to naïve rats that did not have prior experience with nicotine. The beneficial effects of nicotine should not be assumed to occur in individuals with extensive experience with the drug.
Growth factors and their analogs form another class of compounds that hold promise in enhancing recovery after stroke. Thefirst neurotrophic factor to be described was nerve growth factor (NGF), and later Kolb and colleagues showed that it produced about 20% increases in the dendritic arborization and spine density in cortical pyramidal neurons in otherwise normal animals [26]. A subsequent study showed that rats with large cortical strokes had about a 20%
decrease in dendritic arborization in the remaining motor regions and that this loss was completely reversed by NGF [27]. Although the results of this study were compelling, the difficulty with NGF as a potential treatment is that it is expensive and does not pass the blood–brain barrier, a drawback that does not affect FGF-2. FGF-2 holds promise as a potential treat- ment because psychomotor stimulants also transiently increase FGF-2 [28]. There is evidence that adminis- tration of FGF-2 after stroke can stimulate functional improvement, although the effects were small and task-dependent [29]. A later study found that while FGF-2 alone had a minimal effect on recovery from motor cortex injury, FGF-2 was very effective in stim- ulating functional improvement when given in combi- nation with rehabilitation training or complex housing. Furthermore, the functional improvement was correlated with increased synaptogenesis in the remaining motor regions. It may be the case that the endogenous production of neurotrophic factors is potentiated by experience [30]. Thus, it is possible that one mechanism whereby experience facilitates