Michael I. Sandstrom

Genetically engineered mesenchymal stem cells reduce behavioral deficits in the YAC 128 mouse model of Huntington's disease

The purpose of this study was to evaluate the therapeutic effects of the transplantation of bone-marrow mesenchymal stem cells (MSCs), genetically engineered to over-express brain-derived neurotrophic factor (BDNF) or nerve growth factor (NGF) on motor deficits and neurodegeneration in YAC 128 transgenic mice. MSCs, harvested from mouse femurs, were genetically engineered to over-express BDNF and/or NGF and these cells, or the vehicle solution, were injected into the striata of four-month old YAC 128 transgenic and wild-type mice. Assessments of motor ability on the rotarod and the severity of clasping were made one day prior to transplantation and once monthly, thereafter, to determine the effects of the transplanted cells on motor function. The mice were sacrificed at 13-months of age for immunohistological examination. All YAC 128 mice receiving transplants had reduced clasping, relative to vehicle-treated YAC 128 mice, while YAC 128 mice that were transplanted with MSCs which were genetically engineered to over-express BDNF, had the longest latencies on the rotarod and the least amount of neuronal loss within the striatum of the YAC 128 mice. These results indicate that intrastriatal transplantation of MSCs that over-express BDNF may create an environment within the striatum that slows neurodegenerative processes and provides behavioral sparing in the YAC 128 mouse model of HD. Further research on the long-term safety and efficacy of this approach is needed before its potential clinical utility can be comprehensively assessed.

Extracellular ascorbate modulates cortically evoked glutamate dynamics in rat striatum

To determine if extracellular ascorbate, which may increase by several hundred micromolar in striatum during behavioral activation, directly alters glutamate transmission, we monitored striatal glutamate transients evoked by electrical stimulation of cerebral cortex in anesthetized rats tested with varying concentrations of ascorbate (0, 50, 200, and 500 microM) by reverse dialysis. Capillary electrophoresis coupled with laser-induced fluorescence detection was used to analyze dialysates collected at 3-s intervals. Ascorbate elevated striatal glutamate in a concentration-dependent fashion. Addition of 500 microM ascorbate not only more than doubled basal glutamate levels relative to the ascorbate-free condition, but significantly increased both the magnitude of the electrically evoked glutamate response as well as its subsequent return to baseline. In fact, the time required to return to within 10% of the pre-stimulation baseline increased by >100s. Reverse dialysis of iso-ascorbate, in contrast, had no effect on stimulation-evoked glutamate release arguing against an antioxidant effect. It appears, therefore, that the level of extracellular ascorbate plays a critical role in regulating corticostriatal glutamate transmission.

Extracellular ascorbate modulates glutamate dynamics: role of behavioral activation

BackgroundA physiological increase in extracellular ascorbate (AA), an antioxidant vitamin found throughout the striatum, elevates extracellular glutamate (GLU). To determine the role of behavioral arousal in this interaction, microdialysis was used to measure striatal GLU efflux in rats tested in either a lights-off or lights-on condition while reverse dialysis either maintained the concentration of AA at 250 μM or increased it to 1000 μM to approximate endogenous changes.ResultsWhen lights were off, both locomotion and GLU increased regardless of AA dose. In contrast, animals in the lights-on condition were behaviorally inactive, and infusion of 1000, but not 250, μM AA significantly increased extracellular GLU. Interestingly, when ambient light returned to the lights-off group, 1000 μM prolonged the GLU increase relative to the 250 μM group.ConclusionOur results not only support evidence that elevated striatal AA increases extracellular GLU but also indicate that this effect depends on behavioral state and the corresponding level of endogenous GLU release.

Neurotrophic enhancers as therapy for behavioral deficits in rodent models of Huntington's disease: use of gangliosides, substituted pyrimidines, and mesenchymal stem cells.

The interest in using neurotrophic factors as potential treatments for neurodegenerative disorders, such as Huntingtons disease, has grown in the past decade. A major impediment for the clinical utility of neurotrophic factors is their inability to cross the blood-brain barrier in therapeutically significant amounts. Although several novel mechanisms for delivering exogenous neurotrophins to the brain have been developed, most of them involve invasive procedures or present significant risks. One approach to circumventing these problems is using therapeutic agents that can be administered systemically and have the ability to enhance the activity of neurotrophic factors. This review highlights the use of gangliosides, substituted pyrimidines, and mesenchymal stem cells as neurotrophic enhancers that have significant therapeutic potential while avoiding the pitfalls of delivering exogenous neurotrophic factors through the blood-brain barrier. The review focuses on the potential of these neurotrophic enhancers for treating the behavioral deficits in rodent models of Huntingtons disease.

7-nitroindazole attenuates 6-hydroxydopamine-induced spatial learning deficits and dopamine neuron loss in a presymptomatic animal model of Parkinson's disease.

Parkinsons disease (PD) is a neurodegenerative disorder in which loss of dopaminergic (DA) neurons (>50%) in the substantia nigra (SN) precedes most of the overt motor symptoms, making early diagnosis and treatment interventions difficult. Because PD has been associated with free radicals generated by nitric oxide, this study tested whether treatments of 7-nitroindazole (7NI), a nitric-oxide-synthase inhibitor, could reduce cognitive deficits that often emerge before overt motor symptoms in a presymptomatic rat model of PD. Rats were given intraperitoneal injections of 50 mg/kg 7NI (or vehicle) just before receiving bilateral, intrastriatal injections of the DA-toxin, 6-hydroxydopamine (6-OHDA). The rats were then given a battery of motor tasks, and their learning ability was assessed using a spatial reversal task in a water-T maze. Results indicate that 7NI treatments attenuate 6-OHDA-induced spatial learning deficits and protect against DA cell loss in the SN, suggesting that 7NI may have potential as an early, presymptomatic pharmacotherapy for PD.

Early cognitive dysfunction in the HD 51 CAG transgenic rat model of Huntington's disease.

Huntingtons disease (HD) is a neurodegenerative disorder in humans caused by an expansion of a CAG trinucleotide repeat that produces choreic movements, which are preceded by cognitive deficits. The HD transgenic rat (tgHD), which contains the human HD mutation with a 51 CAG repeat allele, exhibits motor deficits that begin when these rats are 12 months of age. However, there are no reports of cognitive dysfunction occurring prior to this. To assess whether cognitive dysfunction might precede motor deficits in tgHD rats, one group of 9-month-old male rats with homozygotic mutated genes and one group of wild-type (WT) rats underwent three testing phases in a unique Spatial Operant Reversal Test (SORT) paradigm, as well as assessment of spontaneous motor activity. After testing, morphological and histological examination of the brains were made. Results indicated that tgHD rats acquired the cued-response (Phase 1) portion of the SORT, but made significantly more errors during the reversal (Phase 2) and during the pseudorandomized reversals (Phase 3) portion of the study, when compared to WT rats. Analysis of the data using mathematical principles of reinforcement revealed no memory, motor, or motivational deficits. These results indicate that early cognitive dysfunction, as measured by the SORT, occur prior to motor deficits, gross anatomical changes, or cell loss in the tgHD rat with 51 CAG repeats, and suggest that this protocol could provide a useful screen for therapeutic studies.

Combinatorial Treatment of Tart Cherry Extract and Essential Fatty Acids Reduces Cognitive Impairments and Inflammation in the mu-p75 Saporin-Induced Mouse Model of Alzheimer's Disease

Alzheimers disease (AD) is a progressive neurodegenerative disorder that affects more than five million Americans and is characterized by a progressive loss of memory, loss of cholinergic neurons in the basal forebrain, formation of amyloid plaques and neurofibrillary tangles, and an increase in oxidative stress. Recent studies indicate that dietary supplements of antioxidants and omega-3 and omega-6 fatty acids may reduce the cognitive deficits in AD patients. The current study tested a combinatorial treatment of antioxidants from tart cherry extract and essential fatty acids from Nordic fish and emu oils for reducing cognitive deficits in the mu-p75 saporin (SAP)-induced mouse model of AD. Mice were given daily gavage treatments of Cerise(®) Total-Body-Rhythm™ (TBR; containing tart cherry extract, Nordic fish oil, and refined emu oil) or vehicle (methylcellulose) for 2 weeks before intracerebroventricular injections of the cholinergic toxin, mu-p75 SAP, or phosphate-buffered saline. The TBR treatments continued for an additional 17 days, when the mice were tested on a battery of cognitive and motor tasks. Results indicate that TBR decreased the SAP-induced cognitive deficits assessed by the object-recognition, place-recognition, and Morris-water-maze tasks. Histological examination of the brain tissue indicated that TBR protected against SAP-induced inflammatory response and loss of cholinergic neurons in the area around the medial septum. These findings indicate that TBR has the potential to serve as an adjunctive treatment which may help reduce the severity of cognitive deficits in disorders involving cholinergic deficits, such as AD.

Bone-marrow-derived mesenchymal stem cells attenuate cognitive deficits in an endothelin-1 rat model of stroke.

PURPOSE Stroke is the third leading cause of death and permanent disability in the United States, often producing long-term cognitive impairments, which are not easily recapitulated in animal models. The goals of this study were to assess whether: (1) the endothelin-1 (ET-1) model of chronic stroke produced discernable cognitive deficits; (2) a spatial operant reversal task (SORT) would accurately measure memory deficits in this model; and (3) bone-marrow-derived mesenchymal stem cells (BMMSCs) could reduce any observed deficits. METHODS Rats were given unilateral intracerebral injections of vehicle or ET-1, a stroke-inducing agent, near the middle cerebral artery. Seven days later, they were given intrastriatal injections of BMMSCs or vehicle, near the ischemic penumbra. The cognitive abilities of the rats were assessed on a novel SORT, which was designed to efficiently distinguish cognitive deficits from potential motoric confounds. RESULTS Rats given ET-1 had significantly more cognitive errors at six weeks post-stroke on the SORT, and that these deficits were attenuated by BMMSC transplants. CONCLUSIONS These findings indicate that: (1) the ET-1 model produces chronic cognitive deficits; (2) the SORT efficiently measures cognitive deficits that are not confounded by motoric impairment; and (3) BMMSCs may be a viable treatment for stroke-induced cognitive dysfunction.

Early Dysfunction of Neural Transmission and Cognitive Processing in Huntington’s Disease

Huntington’s disease (HD) is one of many deteriorative brain diseases, a class of disease in which neurons progressively die. In its final stages, HD robs patients of the dignity of their humanity; denying control of basic movements necessary for communication, facial expression and personal accomplishment. A means to test for the mutation has been available since 1993, when the Huntington’s Disease Collaborative Research Group exposed the huntingtin gene and characterized the nature of the mutation process. Despite this, children of patients often avoid determining their genotype because such a diagnosis is currently merely bleak without hope of remedy, and because of legitimate fears of employment discrimination or difficulties maintaining health insurance given the legal definition of “pre-existing condition.” In the absence of promising treatments or prospects for cures the devastating loss of muscular control during the final stages of disease progression is ominous. It is therefore not uncommon for HD patients to become aware of their own disease rather late into its progression when motor symptoms begin to emerge. As these movement symptoms arise they may be effectively masked by compensatory behavioral strategies. In time, however, these compensatory tactics fail to keep up with the advancing choreic movements which eventually dominate and negate purposeful motor control.

Trans-Differentiation of Rat Mesenchymal Stem Cells into Dopaminergic Neurons for Cell Transplantation

Objective: Transplantation of human embryonic dopaminergic progenitors within the striata of PD patients has provided encouraging results, but ethical concerns and tissue availability limit this approach. The use of mesenchymal stem cells (MSCs) provides a readily available source of cells, as they are derived from adult tissue. This in vitro study explored the use of MSCs as a cell source for DA neuronal induction utilizing a single adenovirus.Methods: Our lab developed a novel adenovirus expressing multiple viral 2A genes allowing for the polycistronic expression of multiple genes (Ascl1, Lmx1a, and Nurr1) for transcription factors that are involved in DA neuron differentiation and used the gene for green fluorescent protein (gfp) to track transfection. MSCs were cultured with the adenovirus, monitored morphological changes as well as expression of gfp as evidenced by fluorescence microscopy. The presence of the viral DNA within the transfected cells was confirmed with PCR, Immunocytochemistry and RTPCR.Results: MSCs cultured with the adenovirus, resulted in morphological changes as well as expression of gfp as evidenced by fluorescence microscopy. The presence of the viral DNA within the transfected cells was confirmed with PCR. Immunocytochemistry and RT-PCR analyses revealed that, cells expressing gfp have nuclear co-labeling of translated transcription factors LMX1a, and NURR1, as well as an up-regulation of these genes, along with an up-regulation of downstream gene targets, such as tyrosine hydroxylase (TH), and the dopamine transporter (DAT).