Preclinical studies of neuroplasticity following experimental brain injury: an update

Abstract

A significant challenge in the care of stroke survivors is chronic functional deficits that limit activities of daily living. Despite substantial functional recovery that can occur in the initial weeks following a stroke, over 50% of stroke survivors exhibit hemiparesis 6 months after stroke, which is particularly significant because motor recovery plateaus after 3–6 months.¹ Because of the compelling need to reduce the burden of these chronic deficits, there is great interest in understanding the specific relationships between neuroplasticity and functional recovery. While fully understanding that these mechanisms will necessarily require clinical research in human cohorts, studies utilizing experimental animal models of brain injury are valuable because preclinical studies provide the ability to induce repeatable lesions, to control the specific environmental exposures that animals receive including the amount of rehabilitative training, to test novel therapies or medications, and to examine the structural and functional changes in cortical organization at different time points. Here we review recent advances that have informed our understanding of neuroplasticity following experimental models of brain injury. Initially, we will review the impact of rehabilitation on functional recovery. Next, we will describe the functional and structural reorganization that is associated with rehabilitation and discuss the functional relevance of specific patterns of reorganization. Finally, we will conclude by describing preclinical evidence for several novel therapies designed to enhance neuroplasticity that span various stages of development towards clinical trials.

The Clinical Relevance of Experimental Models of Brain Injury

One important caveat regarding preclinical models of stroke is that, with few exceptions, these models do not replicate the conditions of human stroke as a clinical entity, and thus suffer to some extent from a lack of face validity. Except in unusual models that are infrequently used, spontaneous strokes simply do not occur in the cohort of otherwise healthy, relatively young laboratory animals utilized in preclinical stroke research. Anecdotally, in three decades of utilizing non-human primates to study the effects of ischemic lesions on sensorimotor function, performing behavioral assessment and post-mortem lesion documentation on over 270 squirrel monkeys, we have not documented a single incident of a spontaneously occurring stroke. We contend that the value of preclinical models of focal ischemia does not stem from face validity, per se. Rather, these studies are valuable for informing human stroke recovery research as they: 1) allow hypothesis-testing regarding basic mechanisms of neuroplasticity and the relationship of plasticity to the capacity for functional recovery, and 2) often demonstrate predictive validity, with motor impairments and recovery profiles that parallel the human recovery trajectory. Nonetheless, extending the predictive ability of such models to the translation of the effects of therapeutic interventions (drugs, devices, behavioral interventions) after ischemic injury remains a significant challenge to the field.

To interpret neuroplasticity following experimental models of brain injury, it is important to consider the implications of the differences between the common animal models used and human stroke survivors. The majority of preclinical studies have used either rodent or non-human primate models. Importantly, both rodents and primates possess primary and secondary cortical motor regions, and substantial efforts have been made to understand functional and structural reorganization in secondary motor areas resulting from damage to primary motor areas. Assuming that secondary reorganization is functionally adaptive, such studies of post-injury cortical plasticity may yield important insights into the capacity of the remaining motor system to respond to therapeutic interventions and support functional recovery. While a growing body of evidence supports neuroplasticity in subcortical brain and spinal cord structures after cortical lesions, the focus of this review is on reorganization in ipsilesional (same side as injury) and contralesional (intact hemisphere) cerebral cortex after focal ischemic lesions. Further, advances in methods for modeling subcortical ischemic lesions (capsular, thalamic, striatal and their combinations) have improved our ability to model more forms of clinical stroke strictly from the standpoint of stroke anatomy. These studies are largely beyond the scope of the present review. Finally, as the vast majority of preclinical studies have focused on the effects of focal ischemic lesions on forelimb function, we limit this review to neuroplasticity in spared cortical areas involved in the control of skilled forelimb function.

Rodents possess two cortical areas associated with the control of skilled forelimb movements. The caudal forelimb area (CFA) is the equivalent of the primate primary motor cortex (M1) and abuts the primary somatosensory forelimb area on its caudal border. A second motor area, the rostral forelimb area (RFA) is located more rostrally, and has been regarded as a premotor area based on similarities in connectivity and neuronal responses associated with movement and pre-movement planning.² While the presence of a secondary premotor region plays a role in the ability of rodents to perform complex grasping movements with the distal forepaw, non-human primates provide a better model of the human motor system with the ability to produce even more complex behaviors. This ability to perform complex movements is facilitated in part by the presence of multiple segregated secondary motor regions including the dorsal and ventral premotor cortices (PMd and PMv), the supplemental motor area (SMA), and the cingulate motor areas (Figure 1).³ Along with the structural and functional organization of cortical motor areas, it is important to consider the roles of the various descending pathways that facilitate motor control. For example, there are interspecies differences among non-human primates in the presence of monosynaptic connections within the corticospinal tract projecting to the ventral horn of the cervical spinal cord, where motoneurons innervating the hand originate.⁴ These structural traits are thought to underlie species-specific differences in distal forelimb dexterity.⁴ Additionally, the rubrospinal and reticulospinal pathways may play a greater role in motor control in non-human primates and rodents compared with humans, and these pathways may provide alternative substrates for recovery of function following experimental brain injuries to a much larger degree than in humans.^5,6 While corticospinal neurons originate from multiple cortical areas, a high concentration are found in M1 and PMv of primates as well as CFA and RFA of rodents.⁷ Taken together, while the clinical implications of experiments using animal models must be considered with care, particularly with regards to distal forelimb function, the presence of multiple distinct cortical areas makes both rodents and non-human primates excellent model species to examine the interaction and reorganization of these regions after brain injury, as might occur after stroke.

Figure 1. Sensorimotor system organization.

Both rodents (left) and non-human primates (right) have multiple differentiated sensory and motor regions. The rodent cortical motor system includes the caudal forelimb area (CFA, rodent M1) and the rostral forelimb area (RFA, rodent premotor cortex). Non-human primates possess multiple premotor areas including the dorsal and ventral premotor cortices (PMd and PMv) the supplementary motor area (SMA) and the cingulate motor areas (CMA). While corticospinal neurons originate in multiple regions, a high concentration of corticospinal neurons is found in M1 and PMv of non-human primates and CFA and RFA of rodents.

Rehabilitation Can Improve Functional Recovery

While a significant number of human stroke survivors are left with chronic motor deficits, rehabilitation has the capacity to improve motor recovery, even in chronic stroke survivors.⁸ Rehabilitative training can also improve motor function following experimental lesions in animal models. For example, after lesion of the internal capsule in rodents, forced use of the impaired forelimb improved motor function with the impaired limb.⁹ Similarly, rehabilitative training, typically consisting of extensive use of the impaired limb for performance of a skilled forelimb task, can improve performance on the trained task in mice and rats.^10–13

In spite of the fact that rehabilitative training can improve function following some experimental ischemic injuries, such training alone is insufficient to restore the ability to perform skilled reaching tasks following more extensive injuries.^14,15 Recent studies have shown that environmental factors, such as being housed in an enriched environment, can improve the effectiveness of rehabilitative training. For example, following large lesions to the CFA in rats (either by an induced ischemic lesion or via a controlled cortical impact), while neither rehabilitative training or housing in an enriched environment alone improved functional recovery relative to control animals, the combination of an enriched environment with rehabilitation did lead to significant improvements in functional recovery.^14,15 In these studies, the enriched environment often includes housing in a larger cage and with multiple animals, as well as access to additional physical items including running wheels or climbing structures.¹⁶ Therefore, the additional improvement associated with housing in an enriched environment could be attributed to a number of adjuvant factors including additional socialization, physical exercise, or cognitive improvements.

While therapies such as rehabilitation can lead to improvements in motor function, different types and time-courses of activity are not necessarily equally beneficial for motor recovery. First, the functional benefits of rehabilitative training are time-dependent. Immediately after the injury, the brain is in a susceptible state and rehabilitative training can lead to decreases in motor function.¹⁷ Further, greatly exaggerated use of the impaired limb within the first week can result in expansion of the injury.¹⁸ Following this very early period, the brain enters a period of enhanced plasticity in which rehabilitation is more effective.¹⁹ Finally, after this critical period of enhanced plasticity ends, there is a more stable, chronic period in which functional recovery is possible but diminished. Interestingly, after this critical period of enhanced rehabilitation has closed, a second ischemic injury distant to the initial injury can reopen this critical window of enhanced plasticity, suggesting that the critical window is specifically tied to the molecular correlates of ischemia.¹⁹ Along with changes in the efficacy of rehabilitation over time, specific behaviors impact the effectiveness of rehabilitative training. For example, increased practice with the non-paretic limb can lead to decreases in the function of the paretic limb and can impede the functional benefits of a later period of training using the paretic limb.²⁰

Cortical Reorganization is Associated with Functional Recovery

Studies demonstrating functional outcomes resulting from behavioral training and environmental enrichment can inform the general mechanisms of cortical plasticity. But it is also important to understand the specific patterns of functional and structural reorganization that occur concurrently with functional recovery and are thought to play a role in mediating this recovery. While the most commonly used experimental stroke model in rodents is the intraluminal suture model of middle cerebral artery occlusion, because of the large and variable size of lesions and functional deficits associated with this model, the majority of studies examining cortical reorganization have utilized focal lesion models which are the focus of our discussion here.²¹

Functional Reorganization of Local and Distant Motor Areas

A number of studies have examined neuroplasticity following experimental lesions by tracking the reorganization of motor regions using intracortical microstimulation (ICMS). Early studies in a non-human primate model of small, focal ischemic lesions showed that while the spared distal forelimb representation in M1 was decreased in size without rehabilitative training, rehabilitation prevented this post-injury loss of distal representation.²² Functional recovery was also associated with enlargements of distal forelimb representations in PMv following lesions made within the distal forelimb region of M1.²³ Larger cortical injuries that included both M1 and PMv resulted in expansion of the SMA distal forelimb representation.²⁴ Along with changes in ICMS maps, increases in activity in local and distant regions has been shown in functional imaging studies. Specifically, following lesions to M1 of macaques made with ibotenic acid, activity in the ipsilesional PMv is increased beginning within 1–2 months after the lesion with increased functional connectivity within the ipsilesional M1 detected at 3–4 months post-lesion.²⁵

In addition to non-human primates, changes in the functional organization of the motor system have also been observed in rodent models. Optogenetic mapping of the motor cortex of the mouse shows displacement of motor representations following small lesions to the primary motor cortex.²⁶ Following larger lesions to CFA (the rodent M1) in the rat, rehabilitation leads to increases in the size of the distal forelimb representation in the ipsilesional RFA (the rodent premotor cortex).^10,11,27 While the majority of studies have focused on examining reorganization within the ipsilesional hemisphere, several studies have also examined changes in the motor representations in the contralesional hemisphere. Following recovery from a cortical lesion, the two hemispheres appear to interact as rehabilitative training with the impaired limb decreases the size of the contralesional CFA relative to healthy controls or lesioned animals that did not receive training.²⁸ On the other hand, maladaptive training with the unimpaired forepaw decreases the size of the perilesional forepaw representation.²⁰ However, the reorganization within the contralesional hemisphere following recovery is also impacted by the size of the lesion. When large and small ischemic lesions were induced in the motor cortex of rodents, increased lesion size correlates with the post-recovery size of RFA in both hemispheres.²⁷ While larger lesions are correlated with larger increases in the size of RFA bilaterally, only the size of the contralesional RFA correlates with the level of functional recovery.²⁷ Taken together, while the optimal motor recovery may be associated with a normalization of motor representations in the ipsilesional hemisphere, the contralesional hemisphere may play an increased role in recovery following larger lesions that cause more substantial motor deficits.

Structural Reorganization of the Motor System

Along with alterations in the functional organization of motor areas, recovery of function has also been associated with structural changes in the same motor regions that likely drive the observed changes in functional organization described above. Following a lesion, axonal sprouting forms new intracortical connections within perilesional areas of the ipsilesional hemisphere as well as new corticospinal and corticorubral connections stemming from both the ipsilesional and contralesional hemispheres.^9,11,29–31 Recent studies have also found that rehabilitation can lead to increased dendritic density within perilesional areas of M1.^13,20,32 When examined using transmission electron microscopy to determine the specific synaptic architecture, it has been found that while both maladaptive and adaptive training can increase the synaptic density in perilesional regions of M1, there were specific differences between the two conditions. Following maladaptive training with the nonparetic limb, increased synaptic density was driven by increases in axodendritic synapses with an increased number of multi-synaptic boutons.²⁰ Following adaptive training, however, the density of perforated synapses which were often associated with perisynaptic astrocytes was increased.³² Therefore, rehabilitation may lead to improved function by encouraging the maturation of appropriate synapses. When considering the changes in the structure of the cortex after a lesion, it is important to recognize that ischemic and mechanical lesions are associated with different types of structural changes.³³ Therefore the specific molecular mechanisms associated with an experimental lesion likely play important roles in the patterns of reorganization that are associated with recovery.

Functional and Structural Reorganization Plays a Causal Role in Recovered Motor Skills

The observation of structural and functional reorganization in animals that experience post-injury motor recovery following rehabilitative training suggests a causal role of both the perilesional M1 and the premotor areas in the recovered motor function. However, the observation of reorganization alone is not sufficient to determine whether these regions play a direct causal role in the recovered motor function. Several studies have used inactivation of the reorganized motor areas to test whether the M1 and ipsilesional premotor cortices are necessary for recovered motor function. First, anisomycin, a protein inhibitor that disrupts synapses, disrupts recovered function when injected into the CFA border region, but not when injected into a non-motor region of rats that had recovered from an ischemic infarct in CFA.³² In contrast, in intact rats, disruption of the central core of CFA with anisomycin impaired performance on a skilled reaching task, but disruption of the CFA border region that was important for motor function in rehabilitated animals had no effect on function.³² Similarly, when an initial lesion is made within CFA followed by a period of rehabilitation, a second lesion to RFA leads to a reinstatement of the initial motor deficits.¹¹ Furthermore, inactivation of PMv in macaques 1–2 months after a lesion to M1 also causes forelimb motor impairments to be reinstated.²⁵ While these studies suggest that the reorganized motor areas play a causal role in the recovered motor movements after recovery has taken place, it is important to note that the observed increases in the size of premotor cortex motor maps are delayed relative to the timing of functional recovery (Figure 2).^10,24 Therefore while secondary motor areas clearly play a causal role in recovered motor function after the recovery has been completed, the time course of the causal role of reorganized motor regions is uncertain.

Figure 2. Motor map reorganization is delayed relative to functional recovery.

Following an ischemic lesion to CFA, the area of the forelimb representation in RFA initially decreases in size with a delayed expansion of RFA (A, red trace).¹⁰ Similarly, following a large lesion to M1, PMd, and PMv of squirrel monkeys, the distal forelimb representation in SMA initially decreases in size with a delayed expansion of the representation (B, red trace).²⁴ While the expansion of motor maps in premotor regions is thought to contribute to motor recovery, the reorganization is delayed relative to the observed improvements in behavioral function (blue traces), raising questions about the specific roles of these secondary motor regions over the course of functional recovery.

Emerging Therapies to Augment Neuroplasticity

Because reorganization of perilesional and distant motor areas plays a causal role in recovered motor function, there has been increasing interest in designing novel therapies to enhance neuroplasticity. While these therapies will ultimately need to be translated to human stroke survivors, tests using experimental models have led to significant advancements of several potential therapies designed to enhance neuroplasticity globally or to strengthen specific intracortical or descending pathways within the motor system.

Open-Loop Brain Stimulation to Enhance Global Plasticity

Several intervention studies in experimental animal models have sought to increase the potential for neuroplasticity in a non-specific way, which when paired with rehabilitation, could lead to improved motor function (Figure 3A). Studies have found that 100Hz epidural cortical stimulation to perilesional areas of rats, when paired with rehabilitation, improves motor recovery relative to rehabilitation alone.³⁴ Importantly, while the functional benefits of epidural stimulation persisted for several months after the therapy, the benefits of cortical stimulation were only observed when started within a few weeks after the initial ischemic lesion.³⁴ Several mechanisms have been proposed for the improvements associated with cortical stimulation including increased neuronal survival and increased neuronal excitability from stimulation-induced depolarization of neurons.³⁵ However, as both cathodal and anodal stimulation have led to functional improvements,³⁵ the specific mechanism of action is uncertain. Because cortical stimulation was effective following cortical but not capsular lesions in rodents, the lesion location and surviving neural structures appear to play important roles in the effectiveness of cortical stimulation.³⁶ Furthermore, negative results with certain stimulation parameters in non-human primate models of stroke and in human clinical trials show that the specific stimulation parameters and lesion characteristics play important roles in the functional benefits of cortical stimulation and that these mechanisms should be better characterized prior to clinical use.^37,38

Figure 3. Emerging technologies to enhance neuroplasticity.

A number of novel technologies have been proposed to enhance and direct neuroplasticity. A. Open-loop interventions seek to non-specifically enhance neuroplasticity to improve the efficacy of rehabilitative training through stimulation of the vagal nerve (left) or direct cortical stimulation (right). Closed-loop strategies seek to drive plasticity in targeted pathways either to strengthen intrinsic cortico-cortical connections (B) or to strengthen the descending motor output from a targeted motor region (C).

Along with direct cortical stimulation, non-invasive stimulation with repetitive transcranial magnetic stimulation (rTMS) or transcranial direct current stimulation (tDCS) have also been proposed as non-invasive methods that may be able to modulate cortical excitability and increase neural plasticity after a cortical injury.³⁹ However, the variety of possible settings tested means that future studies in animal models of brain injury will be necessary to fully characterize the specific mechanisms underlying each of these potential therapies.

Behaviorally-Triggered Stimulation Algorithms

A slightly more targeted approach is to tie stimulation to behavioral events which theoretically would enhance neural plasticity during behaviorally relevant periods. For example, cortical stimulation applied immediately before a pellet retrieval task both normalized low-frequency local-field potentials in the perilesional motor cortex of rodents and improved reaching accuracies in a skilled pellet retrieval task.⁴⁰ In addition to cortical stimulation, vagus nerve stimulation (Figure 3A) has also been proposed to increase cortical plasticity with potential benefits following stroke. When applied after an experimental ischemic lesion, vagus nerve stimulation temporally tied to performance of rehabilitative training leads to improvements in forelimb recovery that generalizes to non-trained tasks,⁴¹ even when delivered in the chronic state.⁴²

Closed-Loop Strategies to Target Specific Pathways

An alternative strategy is to use neuroprosthetic systems to improve recovery by strengthening specific neural pathways. One neuroprosthetic strategy is to strengthen intrinsic connections within the central nervous system by pairing neural activity detected at one location with stimulation applied to a second location (Fig. 3B). This strategy has been demonstrated in a rodent model of TBI. Specifically, following a cortical impact lesion to the CFA, triggering stimulation in S1 based upon the timing of action potentials that were recorded from RFA improves the performance of a skilled pellet retrieval task when compared to either an open-loop sham stimulation protocol or to a control group receiving no stimulation.⁴³ An alternative strategy is to strengthen the extrinsic connections descending from a specific cortical region that facilitate motor output (Figure 3C). This approach has primarily been tested in human subjects using non-invasive systems that detect motor intention with EEG signals to drive an external orthosis or functional electric stimulator.⁴⁴ While rats have demonstrated the ability to control a neuroprosthetic system using neural activity recorded from their perilesional cortex,⁴⁵ few studies have examined the specific mechanisms of neuroplasticity for this type of extrinsic neuroprosthetic system in animal models of brain injury.⁴⁶ However, future studies utilizing animal models of brain injury will be valuable to better understand the specific mechanisms and optimal implementation of these neuroprosthetic systems for neurorehabilitation.

Summary and Future Directions

Experimental models of brain injury have provided valuable tools that have been used to investigate the mechanisms of plasticity that facilitate recovery of lost motor function. Across species and lesion models, the recovery of motor function has been associated with a maintenance or increase in the size of motor representations in both the perilesional portions of M1 and secondary motor areas in the ipsilesional hemisphere. Additionally, an increase and maturation of the synaptic connections in these same regions accompanies the observed functional reorganization. Importantly, the observed reorganization in experimental models corresponds with studies in human subjects that have found that optimal motor recovery is associated with a return to more normal patterns of motor activity.⁴⁷

While preclinical studies allow us to perform well-controlled examinations using repeatable lesions, it is important to note that human stroke survivors demonstrate a more diverse range of stroke locations that do not always correspond well with the most commonly used lesion models.⁴⁸ In particular, the lesion location and extent may be particularly relevant with regard to the role of distant motor areas, including the contralesional hemisphere in motor recovery.²⁷ Furthermore, because this variation in lesion location will impact the success of the translation of therapies into human patients, it will be important to utilize a variety of methods for experimental animals including large and small lesions,²⁷ subcortical lesion locations,⁴⁹ cohorts that include aged animals,¹² and animals with comorbidities similar to those observed in human patients to fully examine the mechanisms of recovery associated with potential therapies. Experimental models of brain injury and ischemia will also continue to be valuable to the future development of novel therapies designed to enhance neuroplasticity. While recent studies have demonstrated the potential of these methods to improve motor function, future studies will be vital to improve our understanding of the specific mechanisms targeted by each intervention, allowing for determination of the optimal implementation for each method, and thereby improving the likelihood of a successful translation into clinical populations.