Andrew W. Grande

Infusion of human umbilical cord blood ameliorates neurologic deficits in rats with hemorrhagic brain injury

Abstract: Umbilical cord blood is a rich source of hematopoietic stem cells. It is routinely used for transplantation to repopulate cells of the immune system. Recent studies, however, have demonstrated that intravenous infusions of umbilical cord blood can ameliorate neurologic deficits associated with ischemic brain injury in rodents. Moreover, the infused cells penetrate into the parenchyma of the brain and adopt phenotypic characteristics typical of neural cells. In the present study we tested the hypothesis that the administration of umbilical cord blood can also diminish neurologic deficits caused by intracerebral hemorrhage (ICH). Intracerebral hemorrhage is a major cause of morbidity and mortality, and at the present time there are no adequate therapies that can minimize the consequences of this cerebrovascular event. ICH was induced in rats by intrastriatal injections of collagenase to cause bleeding in the striatum. Twenty‐four hours after the induction of ICH rats received intravenous saphenous vein infusions of human umbilical cord blood (2.4 × 106 to 3.2 to 106 cells). Animals were evaluated using a battery of tests at day 1 after ICH, but before the administration of umbilical cord blood, and at days 7, and 14 after ICH (days 6 and 13, respectively, after cord blood administration). These tests included a neurological severity test, a stepping test, and an elevated body‐swing test. Animals with umbilical cord blood infusions exhibited significant improvements in (1) the neurologic severity test at 6 and 13 days after cord blood infusion in comparison to saline‐treated animals (P < 0.05); (2) the stepping test at day 6 (P < 0.05); and (3) the elevated body‐swing test at day 13 (P< 0.05). These results demonstrate that the administration of human umbilical cord blood cells can ameliorate neurologic deficits associated with intracerebral hemorrhage.

Neuroprotection by a Bile Acid in an Acute Stroke Model in the Rat

Tauroursodeoxycholic acid (TUDCA), a hydrophilic bile acid, is a strong modulator of apoptosis in both hepatic and nonhepatic cells, and appears to function by inhibiting mitochondrial membrane perturbation. Excitotoxicity, metabolic compromise, and oxidative stress are major determinants of cell death after brain ischemia-reperfusion injury. However, some neurons undergo delayed cell death that is characteristic of apoptosis. Therefore, the authors examined whether TUDCA could reduce the injury associated with acute stroke in a well-characterized model of transient focal cerebral ischemia. Their model of middle cerebral artery occlusion resulted in marked cell death with prominent terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling (TUNEL) within the ischemic penumbra, mitochondrial swelling, and caspase activation. Tauroursodeoxycholic acid administered 1 hour after ischemia resulted in significantly increased bile acid levels in the brain, improved neurologic function, and an approximately 50% reduction in infarct size 2 and 7 days after reperfusion. In addition, TUDCA significantly reduced the number of TUNEL-positive brain cells, mitochondrial swelling, and partially inhibited caspase-3 processing and substrate cleavage. These findings suggest that the mechanism for in vivo neuroprotection by TUDCA is, in part, mediated by inhibition of mitochondrial perturbation and subsequent caspase activation leading to apoptotic cell death. Thus, TUDCA, a clinically safe molecule, may be useful in the treatment of stroke and possibly other apoptosis-associated acute and chronic injuries to the brain.

Environmental impact on direct neuronal reprogramming in vivo in the adult brain

Direct reprogramming of non-neuronal cells to generate new neurons is a promising approach to repair damaged brains. Impact of the in vivo environment on neuronal reprogramming, however, is poorly understood. Here we show that regional differences and injury conditions have significant influence on the efficacy of reprogramming and subsequent survival of newly generated neurons in the adult rodent brain. A combination of local exposure to growth factors and retrovirus-mediated overexpression of the neurogenic transcription factor Neurogenin2 (Neurog2) can induce new neurons from non-neuronal cells in the adult neocortex and striatum where neuronal turnover is otherwise very limited. These two regions respond to growth factors and Neurog2 differently and instruct new neurons to exhibit distinct molecular phenotypes. Moreover, ischemic insult differentially affects differentiation of new neurons in these regions. These results demonstrate strong environmental impact on direct neuronal reprogramming in vivo.

Gsx2 controls region-specific activation of neural stem cells and injury-induced neurogenesis in the adult subventricular zone

Neural stem cells (NSCs) reside in widespread regions along the lateral ventricle and generate diverse olfactory bulb (OB) interneuron subtypes in the adult mouse brain. Molecular mechanisms underlying their regional diversity, however, are not well understood. Here we show that the homeodomain transcription factor Gsx2 plays a crucial role in the region-specific control of adult NSCs in both persistent and injury-induced neurogenesis. In the intact brain, Gsx2 is expressed in a regionally restricted subset of NSCs and promotes the activation and lineage progression of stem cells, thereby controlling the production of selective OB neuron subtypes. Moreover, Gsx2 is ectopically induced in damaged brains outside its normal expression domains and is required for injury-induced neurogenesis in the subventricular zone (SVZ). These results demonstrate that mobilization of adult NSCs is controlled in a region-specific manner and that distinct mechanisms operate in continuous and injury-induced neurogenesis in the adult brain.

Posterior vertebral column subtraction osteotomy: A novel surgical approach for the treatment of multiple recurrences of tethered cord syndrome - Technical note

Recurrent tethered cord syndrome (TCS) has been reported to develop in 5-50% of patients following initial spinal cord detethering operations. Surgery for multiple recurrences of TCS can be difficult and is associated with significant complications. Using a cadaveric tethered spinal cord model, Grande and colleagues demonstrated that shortening of the vertebral column by performing a 15-25-mm thoracolumbar osteotomy significantly reduced spinal cord, lumbosacral nerve root, and terminal filum tension. Based on this cadaveric study, spinal column shortening by a thoracolumbar subtraction osteotomy may be a viable alternative treatment to traditional surgical detethering for multiple recurrences of TCS. In this article, the authors describe the use of posterior vertebral column subtraction osteotomy (PVCSO) for the treatment of 2 patients with multiple recurrences of TCS. Vertebral column resection osteotomy has been widely used in the surgical correction of fixed spinal deformity. The PVCSO is a novel surgical treatment for multiple recurrences of TCS. In such cases, PVCSO may allow surgeons to avoid neural injury by obviating the need for dissection through previously operated sites and may reduce complications related to CSF leakage. The novel use of PVCSO for recurrent TCS is discussed in this report, including surgical considerations and techniques in performing PVCSO.

M1 Muscarinic Receptors Stimulate Rapid and Prolonged Phases of Neuronal Nitric Oxide Synthase Activity: Involvement of Different Calcium Pools

Abstract: This study shows that activation of M1 muscarinic receptors, when coexpressed in Chinese hamster ovary (CHO)‐K1 cells with neuronal nitric oxide (NO) synthase (nNOS), produces early and late phases of elevation of both intracellular Ca2+ concentration and nNOS activity. We examined the relationship between receptor‐mediated increases in intracellular Ca2+ concentration and activation of nNOS over both short and long intervals using guanosine 3′,5′‐cyclic monophosphate (cGMP) formation as a measure of nNOS activity. The rapid phase of nNOS activation was dependent on release of Ca2+ from intracellular stores in both the CHO M1/nNOS transfected cells and in neuroblastoma (N1E‐115) cells, in which muscarinic receptors and nNOS are endogenously expressed. Two single point mutations in the M1 muscarinic receptor that have previously been shown to uncouple differentially the receptor from phosphoinositide hydrolysis produced parallel attenuation of the rapid phase of nNOS activation. Characterization of the prolonged phase of nNOS activation was done using the conversion of l‐[3H]arginine to l‐[3H]citrulline as well as cGMP formation following stimulation of M1 muscarinic receptors for 60 min. Both responses were dependent on influx of extracellular Ca2+ and were accompanied by prolonged formation of NO at functionally effective levels as late as 60 min following receptor activation. Therefore, this study demonstrates for the first time the existence of two mechanistically distinct phases of nNOS activation that are dependent on different sources of Ca2+.

Stroke Recovery and Rehabilitation Research: Issues, Opportunities, and the National Institutes of Health StrokeNet

Stroke is the second leading cause of death and the third leading cause of disability-adjusted life years worldwide. Although numerous therapies have been developed over the past 10 years to treat acute ischemic stroke, the stark reality remains that only 5% of these patients are so treated in the United States,1 in part, because of treatment window times <3 to 6 hours post-onset, and many of these 5% nonetheless have significant long-term disability. Acute treatment options after hemorrhagic stroke remain limited.2 In parallel with efforts to further develop acute stroke interventions, researchers are studying recovery and rehabilitation treatments, which can have a treatment time window measured in days, weeks, or months poststroke. To achieve this goal, therapies aim to maximize function in brain areas that survive the stroke or provide compensatory approaches to improve overall function. Strategies targeting recovery and rehabilitation must be seen as distinct from acute stroke therapies, such as reperfusion or neuroprotection, where the strategy is to limit the severity of ischemic injury, including preserving penumbral tissue and reducing infarct size. Preclinical and translational research have successfully identified numerous molecular and physiological events spontaneously arising in the nervous system during the days-to-weeks after an infarct, and, subsequently, potential restorative therapies that target these events to improve long-term behavioral outcomes.3,4 In parallel, a burgeoning volume of data from human subjects has emerged regarding mechanisms of recovery from stroke. Together, these efforts inform translation into clinical studies for several classes of therapy, including small molecules, growth factors, stem cells, monoclonal antibodies, brain stimulation, robotics and other devices, cognitive strategies, intensive training, and telerehabilitation.5,6 The majority of patients with stroke survive the initial event but go on to live with significant disability for many years. Indeed, there are >7 million stroke survivors …

Neural Repair and Neuroprotection with Stem Cells in Ischemic Stroke

Stem cells have been touted as a potential source of cells for repair in regenerative medicine. When transplanted into the central nervous system, stem cells have been shown to differentiate into neurons and glia. Recent studies, however, have also revealed neuroprotective properties of stem cells. These studies suggest that various types of stem cells are able to protect against the loss of neurons in conditions of ischemic brain injury. In this article, we discuss the use of stem cells for ischemic stroke and the parameters under which neuroprotection can occur in the translation of stem cell therapy to the clinical setting.

Revisiting neural stem cell identity

The discovery of neural stem cells (NSCs) in the adult mammalian central nervous system (CNS) has dramatically changed our view on the regenerative capacity of this organ (1, 2). We now realize that the adult brain, including humans, retains the ability to replenish its cellular constituents, neurons and glia, although the extent is very limited compared with lower vertebrates (2). Cell turnover driven by NSCs has been implicated in higher brain functions such as learning and memory (3). Adult NSCs have also been shown to participate in neuronal cell replacement after injury, raising the possibility of stem cell-based therapy for neurological disorders (4). Despite extensive studies in the past decade, one fundamental question remains unanswered: What is the identity of NSCs? In 1999, two groups reported apparently contradictory results: Johansson et al. (5) provided evidence that ependymal cells, which constitute a ciliated single-cell-thick epithelial layer lining the lateral ventricle (LV), retain the characteristics of NSCs (Fig. 1 A). In contrast, Doetsch et al. (6) identified glial fibrillary acidic protein (GFAP)-positive astrocyte-like cells, which reside in a region beneath the ependymal layer called the subependymal layer or subventricular zone (SVZ), as NSCs. Although these two results were not necessarily mutually exclusive, they sparked a debate that has been ongoing ever since. For better understanding of the basic biology of NSCs and their future successful use for therapy, reconciliation of this long-debated issue is awaited. The work of Coskun et al. (7) in this issue of PNAS addressed this question by using new tools and approaches.

Amelioration of ischemic brain injury in rats with human umbilical cord blood stem cells: Mechanisms of action

Despite the high prevalence and devastating outcome, there remain a few options for treatment of ischemic stroke. Currently available treatments are limited by a short time window for treatment and marginal efficacy when used. We have tested a human umbilical cord blood-derived stem cell line that has been shown to result in a significant reduction in stroke infarct volume as well as improved functional recovery following stroke in the rat. In the present study we address the mechanism of action and compared the therapeutic efficacy of high- versus low-passage nonhematopoietic umbilical cord blood stem cells (nh-UCBSCs). Using the middle cerebral arterial occlusion (MCAo) model of stroke in Sprague–Dawley rats, we administered nh-UCBSC by intravenous (IV) injection 2 days following stroke induction. These human cells were injected into rats without any immune suppression, and no adverse reactions were detected. Both behavioral and histological analyses have shown that the administration of these cells reduces the infarct volume by 50% as well as improves the functional outcome of these rats following stroke for both high- and low-passaged nh-UCBSCs. Flow cytometry analysis of immune cells present in the brains of normal rats, rats with ischemic brain injury, and ischemic animals with nh-UCBSC treatment confirmed infiltration of macrophages and T cells consequent to ischemia and reduction to normal levels with nh-UCBSC treatment. Flow cytometry also revealed a restoration of normal levels of microglia in the brain following treatment. These data suggest that nh-UCBSCs may act by inhibiting immune cell migration into the brain from the periphery and possibly by inhibition of immune cell activation within the brain. nh-UCBSCs exhibit great potential for treatment of stroke, including the fact that they are associated with an increased therapeutic time window, no known ill-effects, and that they can be expanded to high numbers for, and stored for, treatment.