Elena V. Zoubina

Extensive Cortical Rewiring after Brain Injury

Previously, we showed that the ventral premotor cortex (PMv) underwent neurophysiological remodeling after injury to the primary motor cortex (M1). In the present study, we examined cortical connections of PMv after such lesions. The neuroanatomical tract tracer biotinylated dextran amine was injected into the PMv hand area at least 5 months after ischemic injury to the M1 hand area. Comparison of labeling patterns between experimental and control animals demonstrated extensive proliferation of novel PMv terminal fields and the appearance of retrogradely labeled cell bodies within area 1/2 of the primary somatosensory cortex after M1 injury. Furthermore, evidence was found for alterations in the trajectory of PMv intracortical axons near the site of the lesion. The results suggest that M1 injury results in axonal sprouting near the ischemic injury and the establishment of novel connections within a distant target. These results support the hypothesis that, after a cortical injury, such as occurs after stroke, cortical areas distant from the injury undergo major neuroanatomical reorganization. Our results reveal an extraordinary anatomical rewiring capacity in the adult CNS after injury that may potentially play a role in recovery.

Post-infarct cortical plasticity and behavioral recovery using concurrent cortical stimulation and rehabilitative training: A feasibility study in primates

Abstract Stroke is often characterized by incomplete recovery and chronic motor impairments. A nonhuman primate model of cortical ischemia was used to evaluate the feasibility of using device-assisted cortical stimulation combined with rehabilitative training to enhance behavioral recovery and cortical plasticity. Following preinfarct training on a unimanual motor task, maps of movement representations in primary motor cortex were derived. Then, an ischemic infarct was produced which destroyed the hand representation. Several weeks later, a second cortical map was derived to guide implantation of a surface electrode over periinfarct motor cortex. After several months of spontaneous recovery, monkeys underwent subthreshold electrical stimulation combined with rehabilitative training for several weeks. Post-therapy behavioral performance was tracked for several additional months. A third cortical map was derived several weeks post-therapy to examine changes in motor representations. Monkeys showed significant improvements in motor performance (success, speed, and efficiency) following therapy, which persisted for several months. Cortical mapping revealed large-scale emergence of new hand representations in peri-infarct motor cortex, primarily in cortical tissue underlying the electrode. Results support the feasibility of using a therapy approach combining peri-infarct electrical stimulation with rehabilitative training to alleviate chronic motor deficits and promote recovery from cortical ischemic injury.

VEGF protein associates to neurons in remote regions following cortical infarct.

Vascular endothelial growth factor (VEGF) is thought to contribute to both neuroprotection and angiogenesis after stroke. While increased expression of VEGF has been demonstrated in animal models after experimental ischemia, these studies have focused almost exclusively on the infarct and peri-infarct regions. The present study investigated the association of VEGF to neurons in remote cortical areas at three days after an infarct in primary motor cortex (M1). Although these remote areas are outside of the direct influence of the ischemic injury, remote plasticity has been implicated in recovery of function. For this study, intracortical microstimulation techniques identified primary and premotor cortical areas in a non-human primate. A focal ischemic infarct was induced in the M1 hand representation, and neurons and VEGF protein were identified using immunohistochemical procedures. Stereological techniques quantitatively assessed neuronal-VEGF association in the infarct and peri-infarct regions, M1 hindlimb, M1 orofacial, and ventral premotor hand representations, as well as non-motor control regions. The results indicate that VEGF protein significantly increased association to neurons in specific remote cortical areas outside of the infarct and peri-infarct regions. The increased association of VEGF to neurons was restricted to cortical areas that are functionally and/or behaviorally related to the area of infarct. There was no significant increase in M1 orofacial region or in non-motor control regions. We hypothesize that enhancement of neuronal VEGF in these functionally related remote cortical areas may be involved in recovery of function after stroke, through either neuroprotection or the induction of remote angiogenesis.

Expression of estrogen receptors α and β by sympathetic ganglion neurons projecting to the proximal urethra of female rats

ABSTRACTPurpose: Urinary incontinence is prevalent in postmenopausal women and estrogen is commonly administered therapeutically. In animal models estrogen increases urethral smooth muscle agonist induced contraction but a consistent clinical benefit in humans has not been confirmed. A reason may be that estrogen affects tissues other than the urethra that are involved in continence. We determined if sympathetic nerves projecting to the urethra may also be a target for estrogen.Materials and Methods: Sympathetic neurons innervating proximal urethra smooth muscle were identified by injection of the retrograde tracer Fast Blue (Dr. Illing GmbH and Co. KG, Gross-Umstadt, Germany) in 10 ovariectomized adult female rats. Rats received a single injection of 10 μg./kg. estradiol benzoate or vehicle 24 hours before tissue harvest. Retrograde labeled sympathetic neurons expressing estrogen receptors α and β in prevertebral and paravertebral ganglia were identified by immunostaining.Results: Approximately 80% of Fa...

Axonal degeneration and regeneration in rat uterus during the estrous cycle.

Uterine innervation of the adult virgin rat changes throughout the estrous cycle. Nerves immunoreactive for the pan-neuronal marker protein gene product 9.5 and the sympathetic marker dopamine beta-hydroxylase are maximal at diestrus and minimal at estrus, whereas presumptive sensory and parasympathetic axons are unchanged. In the present study, we used quantitative electron microscopy to determine if depletion of immunoreactive nerves from the myometrium is due to loss of structurally intact axons, and whether this occurs through degeneration or retraction. Numbers of intact myometrial axons per unit sectional area were greatest at diestrus and least at estrus, while myometrial area was smallest at diestrus and greatest at estrus. However, depletion of intact axons at estrus was evident even after correcting for changes in uterine size. Varicosities adjacent to smooth muscle cells did not vary significantly with respect to their ultrastructural features or distance to the nearest smooth muscle target cell. Because retracting axons show increases in organelle content and distances to target cells, retraction probably does not play a major role in reducing uterine innervation. In contrast, axons with ultrastructural features consistent with degeneration (organelle and axolemmal disintegration, abnormal electron opacity, dense inclusion bodies) were significantly increased at proestrus and estrus. Growth cones were observed only at metestrus and diestrus. We conclude that cyclical degeneration and regeneration of myometrial innervation is a normal feature of the virgin adult rat.

A Single Injection of d-Amphetamine Facilitates Improvements in Motor Training Following a Focal Cortical Infarct in Squirrel Monkeys

Background. There is growing interest in the use of d-amphetamine (d-AMPH) as a pharmacological treatment to supplement rehabilitative therapy following stroke. Based on the success of earlier animal models, several clinical studies have demonstrated beneficial effects of applying physical rehabilitation while stroke patients are under the influence of d-AMPH. To begin to understand the neural mechanisms underlying this promising adjuvant therapy, the authors examined the effects of a single pairing of d-AMPH and rehabilitative training on motor performance after cortical infarct in squirrel monkeys. Methods. Microelectrode stimulation techniques were used to delineate hand movement areas in the primary motor cortex prior to delivering a unilateral infarct to the complete hand representation. Postinfarct recovery was assessed for 3 groups of monkeys: d-AMPH + training, saline + training, and spontaneous recovery (SR). Postinfarct training groups received 14 consecutive days of motor skill training on a reach and retrieval task. A single injection of d-AMPH (0.25 mg/kg) or saline was given only on the 1st day of training (postinfarct day 10). Monkeys in the SR group had only minimal exposure to the training task once per week to monitor recovery. Results. The results show that a single coupling of d-AMPH + training initiated 10 days after cortical infarct facilitated the rate of recovery and improved performance (68% improvement from 1st day of training) beyond the level achieved by the monkeys in the saline + training group (27% improved from 1st day of training). Conclusions. D-AMPH is a potent modulator of behavioral recovery following an ischemic infarct in nonhuman primates.

Neuronal HIF-1α protein and VEGFR-2 immunoreactivity in functionally related motor areas following a focal M1 infarct

Clinical and experimental data support a role for the intact cortex in recovery of function after stroke, particularly ipsilesional areas interconnected to the infarct. There is, however, little understanding of molecular events in the intact cortex, as most studies focus on the infarct and peri-infarct regions. This study investigated neuronal immunoreactivity for hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2) in remote cortical areas 3 days after a focal ischemic infarct, as both HIF-1α and VEGFR-2 have been implicated in peri-infarct neuroprotection. For this study, intracortical microstimulation techniques defined primary motor (M1) and premotor areas in squirrel monkeys (genus Saimiri). An infarct was induced in the M1 hand representation, and immunohistochemical techniques identified neurons, HIF-1α and VEGFR-2. Stereologic techniques quantified the total neuronal populations and the neurons immunoreactive for HIF-1α or VEGFR-2. The results indicate that HIF-1α upregulation is confined to the infarct and peri-infarct regions. Increases in VEGFR-2 immunoreactivity occurred; however, in two remote regions: the ventral premotor hand representation and the M1 hindlimb representation. Neurons in these representations were previously shown to undergo significant increases in VEGF protein immunoreactivity, and comparison of the two data sets showed a significant correlation between levels of VEGF and VEGFR-2 immunoreactivity. Thus, while remote areas undergo a molecular response to the infarct, we hypothesize that there is a delay in the initiation of the response, which ultimately may increase the ‘window of opportunity’ for neuroprotective interventions in the intact cortex.

Effects of a Rostral Motor Cortex Lesion on Primary Motor Cortex Hand Representation Topography in Primates

Background. Small lesions to rostral versus caudal portions of the hand representation in the primary motor cortex (M1) produce different behavioral deficits. The goal of the present study was to determine if rehabilitative training has similar effects on functional topography of the spared M1 after rostral versus previously reported caudal M1 lesions. Methods. Following a lesion to the rostral M1 hand area, monkeys were trained for 1 h/day for 30 days to retrieve food pellets from small wells using their impaired hand. Electrophysiological maps of the M1 were derived in anesthetized monkeys before infarct and after rehabilitative training using intracortical microstimulation. Results. After a lesion to the rostral M1 and rehabilitative training, the size of the spared hand representation decreased 1.2%. This change is not statistically different from the 9% increase seen after caudal M1 lesion and rehabilitative training (P > 0.2). Conclusion. Postlesion training spares peri-infarct hand area regardless of whether the lesion is in the rostral or caudal M1.

Effects of Postinfarct Myelin-Associated Glycoprotein Antibody Treatment on Motor Recovery and Motor Map Plasticity in Squirrel Monkeys

Background and Purpose— New insights into the brain’s ability to reorganize after injury are beginning to suggest novel restorative therapy targets. Potential therapies include pharmacological agents designed to promote axonal growth. The purpose of this study was to test the efficacy of one such drug, GSK249320, a monoclonal antibody that blocks the axon outgrowth inhibition molecule, myelin-associated glycoprotein, to facilitate recovery of motor skills in a nonhuman primate model of ischemic cortical damage. Methods— Using a between-groups repeated-measures design, squirrel monkeys were randomized to 1 of 2 groups: an experimental group received intravenous GSK249320 beginning 24 hours after an ischemic infarct in motor cortex with repeated dosages given at 1-week intervals for 6 weeks and a control group received only the vehicle at matched time periods. The primary end point was a motor performance index based on a distal forelimb reach-and-retrieval task. Neurophysiological mapping techniques were used to determine changes in spared motor representations. Results— All monkeys recovered to baseline motor performance levels by postinfarct day 16. Functional recovery in the experimental group was significantly facilitated on the primary end point, albeit using slower movements. At 7 weeks post infarct, motor maps in the spared ventral premotor cortex in the experimental group decreased in area compared with the control group. Conclusions— GSK249320, initiated 24 hours after a focal cortical ischemic infarct, facilitated functional recovery. Together with the neurophysiological data, these results suggest that GSK249320 has a substantial biological effect on spared cortical tissue. However, its mechanisms of action may be widespread and not strictly limited to peri-infarct cortex and nearby premotor areas.

Effects of Subdural Monopolar Cortical Stimulation Paired With Rehabilitative Training on Behavioral and Neurophysiological Recovery After Cortical Ischemic Stroke in Adult Squirrel Monkeys.

Background. Cortical stimulation (CS) combined with rehabilitative training (RT) has proven effective for enhancing poststroke functional recovery in rats, but human clinical trials have had mixed outcomes. Objective. To assess the efficacy of CS/RT versus RT in a nonhuman primate model of cortical ischemic stroke. Methods. Squirrel monkeys learned a pellet retrieval task, then received an infarct to the distal forelimb (DFL) representation of primary motor cortex. A subdural monopolar electrode was implanted over the spared DFL representation in dorsal premotor cortex (PMD). Seven weeks postinfarct, monkeys underwent 4 to 6 weeks of RT (n = 8) or CS/RT (n = 7; 100 Hz, cathodal current) therapy. Behavioral performance was assessed before and after infarct, prior to therapy, and 1 and 12 weeks posttherapy (follow-up). The primary outcome measure was motor performance at 1 week posttherapy. Secondary outcomes included follow-up performance at 12 weeks and treatment-related changes in neurophysiological maps of spared DFL representations. Results. While postinfarct performance deficits were found in all monkeys, both groups demonstrated similar recovery profiles, with no difference in motor recovery between the RT and CS/RT groups. Posttherapy, PMD DFL area was significantly expanded in the RT group but not the CS/RT group. A significant relationship was found between motor recovery and DFL expansion in premotor cortex. Conclusions. Results suggest that the specific parameters utilized here were not optimal for promoting behavioral recovery in nonhuman primates. Though CS/RT has consistently shown efficacy in rat stroke models, the present finding has cautionary implications for translation of CS/RT therapy to clinical populations.