Probing sporadic and familial Alzheimer's disease using induced pluripotent stem cells.

Our understanding of Alzheimer's disease pathogenesis is currently limited by difficulties in obtaining live neurons from patients and the inability to model the sporadic form of the disease. It may be possible to overcome these challenges by reprogramming primary cells from patients into induced pluripotent stem cells (iPSCs). Here we reprogrammed primary fibroblasts from two patients with familial Alzheimer's disease, both caused by a duplication of the amyloid-ß precursor protein gene (APP; termed APP(Dp)), two with sporadic Alzheimer's disease (termed sAD1, sAD2) and two non-demented control individuals into iPSC lines. Neurons from differentiated cultures were purified with fluorescence-activated cell sorting and characterized. Purified cultures contained more than 90% neurons, clustered with fetal brain messenger RNA samples by microarray criteria, and could form functional synaptic contacts. Virtually all cells exhibited normal electrophysiological activity. Relative to controls, iPSC-derived, purified neurons from the two APP(Dp) patients and patient sAD2 exhibited significantly higher levels of the pathological markers amyloid-ß(1-40), phospho-tau(Thr 231) and active glycogen synthase kinase-3ß (aGSK-3ß). Neurons from APP(Dp) and sAD2 patients also accumulated large RAB5-positive early endosomes compared to controls. Treatment of purified neurons with ß-secretase inhibitors, but not γ-secretase inhibitors, caused significant reductions in phospho-Tau(Thr 231) and aGSK-3ß levels. These results suggest a direct relationship between APP proteolytic processing, but not amyloid-ß, in GSK-3ß activation and tau phosphorylation in human neurons. Additionally, we observed that neurons with the genome of one sAD patient exhibited the phenotypes seen in familial Alzheimer's disease samples. More generally, we demonstrate that iPSC technology can be used to observe phenotypes relevant to Alzheimer's disease, even though it can take decades for overt disease to manifest in patients.

The Rheb-mTOR pathway is upregulated in reactive astrocytes of the injured spinal cord.

Astrocytes in the CNS respond to tissue damage by becoming reactive. They migrate, undergo hypertrophy, and form a glial scar that inhibits axon regeneration. Therefore, limiting astrocytic responses represents a potential therapeutic strategy to improve functional recovery. It was recently shown that the epidermal growth factor (EGF) receptor is upregulated in astrocytes after injury and promotes their transformation into reactive astrocytes. Furthermore, EGF receptor inhibitors were shown to enhance axon regeneration in the injured optic nerve and promote recovery after spinal cord injury. However, the signaling pathways involved were not elucidated. Here we show that in cultures of adult spinal cord astrocytes EGF activates the mTOR pathway, a key regulator of astrocyte physiology. This occurs through Akt-mediated phosphorylation of the GTPase-activating protein Tuberin, which inhibits Tuberin's ability to inactivate the small GTPase Rheb. Indeed, we found that Rheb is required for EGF-dependent mTOR activation in spinal cord astrocytes, whereas the Ras-MAP kinase pathway does not appear to be involved. Moreover, astrocyte growth and EGF-dependent chemoattraction were inhibited by the mTOR-selective drug rapamycin. We also detected elevated levels of activated EGF receptor and mTOR signaling in reactive astrocytes in vivo in an ischemic model of spinal cord injury. Furthermore, increased Rheb expression likely contributes to mTOR activation in the injured spinal cord. Interestingly, injured rats treated with rapamycin showed reduced signs of reactive gliosis, suggesting that rapamycin could be used to harness astrocytic responses in the damaged nervous system to promote an environment more permissive to axon regeneration.

Stem cells: comprehensive treatments for amyotrophic lateral sclerosis in conjunction with growth factor delivery.

Amyotrophic lateral sclerosis (ALS) is characterized by loss of both upper and lower motor neurons. ALS progression is complex and likely due to cellular dysfunction at multiple levels, including mitochondrial dysfunction, glutamate excitotoxicity, oxidative stress, axonal dysfunction, reactive astrocytosis, and mutant superoxide dismutase expression, therefore, treatment must provide neuronal protection from multiple insults. A significant amount of ALS research focuses on growth factor-based therapies. Growth factors including insulin-like growth factor-I, vascular endothelial growth factor, brain-derived neurotrophic factor, and glial-derived neurotrophic factor exhibit robust neuroprotective effects on motor neurons in ALS models. Issues concerning growth factor delivery, stability and unwanted side effects slow the transfer of these treatments to human ALS patients. Stem cells represent a new therapeutic approach offering both cellular replacement and trophic support for the existing population. Combination therapy consisting of stem cells expressing beneficial growth factors may provide a comprehensive treatment for ALS.

Spinal p38 mitogen-activated protein kinase mediates allodynia induced by first-degree burn in the rat.

Activation of p38 mitogen-activated protein kinase (MAPK) in the spinal cord has been implicated in the development and maintenance of pain states. In this study, we tested whether p38 MAPK is involved in the response to first-degree burn of the hind paw. This injury induces central sensitization leading to tactile allodynia and is mediated by activation of Ca(2+) permeable AMPA/kainate receptors through PKC and PKA. We demonstrate that p38 MAPK is rapidly and robustly activated in the superficial spinal dorsal horn after mild thermal injury to the hind paw. Activated p38 MAPK was localized primarily to microglia and to a lesser extent in oligodendrocytes and lamina II neurons. Astrocytes were not involved in the p38 MAPK response. Intrathecal pretreatment of pharmacological inhibitors of p38 MAPK (SB203580, SD-282) dose-dependently blocked development of tactile allodynia, a characteristic of the first-degree burn model. The effects of the inhibitors on tactile allodynia were lost when they were administered after injury. These studies identify p38 MAPK as a major mediator of tactile allodynia, most likely activated downstream of AMPA/kainate receptors.

Spinal astrocyte glutamate receptor 1 overexpression after ischemic insult facilitates behavioral signs of spasticity and rigidity.

Using a rat model of ischemic paraplegia, we examined the expression of spinal AMPA receptors and their role in mediating spasticity and rigidity. Spinal ischemia was induced by transient occlusion of the descending aorta combined with systemic hypotension. Spasticity/rigidity were identified by simultaneous measurements of peripheral muscle resistance (PMR) and electromyography (EMG) before and during ankle flexion. In addition, Hoffman reflex (H-reflex) and motor evoked potentials (MEPs) were recorded from the gastrocnemius muscle. Animals were implanted with intrathecal catheters for drug delivery and injected with the AMPA receptor antagonist NGX424 (tezampanel), glutamate receptor 1 (GluR1) antisense, or vehicle. Where intrathecal vehicle had no effect, intrathecal NGX424 produced a dose-dependent suppression of PMR [ED50 of 0.44 microg (0.33-0.58)], as well as tonic and ankle flexion-evoked EMG activity. Similar suppression of MEP and H-reflex were also seen. Western blot analyses of lumbar spinal cord tissue from spastic animals showed a significant increase in GluR1 but decreased GluR2 and GluR4 proteins. Confocal and electron microscopic analyses of spinal cord sections from spastic animals revealed increased GluR1 immunoreactivity in reactive astrocytes. Selective GluR1 knockdown by intrathecal antisense treatment resulted in a potent reduction of spasticiy and rigidity and concurrent downregulation of neuronal/astrocytic GluR1 in the lumbar spinal cord. Treatment of rat astrocyte cultures with AMPA led to dose-dependent glutamate release, an effect blocked by NGX424. These data suggest that an AMPA/kainate receptor antagonist can represent a novel therapy in modulating spasticity/rigidity of spinal origin and that astrocytes may be a potential target for such treatment.

Survival of syngeneic and allogeneic iPSC-derived neural precursors after spinal grafting in minipigs.

The use of autologous (or syngeneic) cells derived from induced pluripotent stem cells (iPSCs) holds great promise for future clinical use in a wide range of diseases and injuries. It is expected that cell replacement therapies using autologous cells would forego the need for immunosuppression, otherwise required in allogeneic transplantations. However, recent studies have shown the unexpected immune rejection of undifferentiated autologous mouse iPSCs after transplantation. Whether similar immunogenic properties are maintained in iPSC-derived lineage-committed cells (such as neural precursors) is relatively unknown. We demonstrate that syngeneic porcine iPSC-derived neural precursor cell (NPC) transplantation to the spinal cord in the absence of immunosuppression is associated with long-term survival and neuronal and glial differentiation. No tumor formation was noted. Similar cell engraftment and differentiation were shown in spinally injured transiently immunosuppressed swine leukocyte antigen (SLA)-mismatched allogeneic pigs. These data demonstrate that iPSC-NPCs can be grafted into syngeneic recipients in the absence of immunosuppression and that temporary immunosuppression is sufficient to induce long-term immune tolerance after NPC engraftment into spinally injured allogeneic recipients. Collectively, our results show that iPSC-NPCs represent an alternative source of transplantable NPCs for the treatment of a variety of disorders affecting the spinal cord, including trauma, ischemia, or amyotrophic lateral sclerosis.

Low-speed subcellular fractionation method for determining noxious stimulus-evoked spinal neurokinin-1 receptor internalization.

Substance P release from nociceptive primary afferents activates post-synaptic neurokinin-1 (NK-1) receptors causing subsequent NK-1 receptor internalization. Fluorescent immunohistochemistry is typically used to quantify NK-1 receptor internalization, an indirect measure of substance P (SP) release. However, this technique entails several limitations that restrict its application. Using simple subcellular fractionation and immunoblotting methods, we demonstrate that intrathecal SP invokes a rapid and dose-dependent increase in dorsal horn cytoplasmic NK-1 receptors. We also show that hind paw compression and noxious thermal stimulation increase cytoplasmic NK-1 receptor, when compared to sham stimulations. Fluorescent immunohistochemistry confirmed that increases in cytoplasmic NK-1 corresponded with increased NK-1 receptor internalization. Herein, we report that low-speed centrifugation and Western immunoblotting provide NK-1 internalization results consistent with those obtained by more traditional methods. These data support previous findings demonstrating a role for spinal NK-1 receptors in nociceptive processing.

Amelioration of Penetrating Ballistic-Like Brain Injury Induced Cognitive Deficits after Neuronal Differentiation of Transplanted Human Neural Stem Cells.

Penetrating traumatic brain injury (PTBI) is one of the major cause of death and disability worldwide. Previous studies with penetrating ballistic-like brain injury (PBBI), a PTBI rat model revealed widespread perilesional neurodegeneration, similar to that seen in humans following gunshot wound to the head, which is unmitigated by any available therapies to date. Therefore, we evaluated human neural stem cell (hNSC) engraftment to putatively exploit the potential of cell therapy that has been seen in other central nervous system injury models. Toward this objective, green fluorescent protein (GFP) labeled hNSC (400,000 per animal) were transplanted in immunosuppressed Sprague-Dawley (SD), Fisher, and athymic (ATN) PBBI rats 1 week after injury. Tacrolimus (3 mg/kg 2 days prior to transplantation, then 1 mg/kg/day), methylprednisolone (10 mg/kg on the day of transplant, 1 mg/kg/week thereafter), and mycophenolate mofetil (30 mg/kg/day) for 7 days following transplantation were used to confer immunosuppression. Engraftment in SD and ATN was comparable at 8 weeks post-transplantation. Evaluation of hNSC differentiation and distribution revealed increased neuronal differentiation of transplanted cells with time. At 16 weeks post-transplantation, neither cell proliferation nor glial lineage markers were detected. Transplanted cell morphology was similar to that of neighboring host neurons, and there was relatively little migration of cells from the peritransplant site. By 16 weeks, GFP-positive processes extended both rostrocaudally and bilaterally into parenchyma, spreading along host white matter tracts, traversing the internal capsule, and extending ∼13 mm caudally from transplantation site reaching into the brainstem. In a Morris water maze test at 8 weeks post-transplantation, animals with transplants had shorter latency to platform than vehicle-treated animals. However, weak injury-induced cognitive deficits in the control group at the delayed time point confounded benefits of durable engraftment and neuronal differentiation. Therefore, these results justify further studies to progress towards clinical translation of hNSC therapy for PTBI.

Development of baclofen tolerance in a rat model of chronic spasticity and rigidity.

Systemic or spinal treatment with baclofen has been associated with the development of tolerance in patients with chronic spasticity. In the present study, we used a rat model of spinal ischemia-induced spasticity to characterize the development of baclofen tolerance after chronic intrathecal (i.t.) baclofen infusion. Following the induction of spinal ischemia and the development of behavioral spasticity, animals were implanted with i.t. catheters connected to osmotic pumps to continuously infuse baclofen (1.0 microg/0.5 microl/h). Hindleg peripheral muscle resistance (PMR) was measured periodically after initiation of chronic infusion and after bolus i.t. baclofen injection (1.0 microg). Peripheral muscle resistance was significantly decreased at the onset of baclofen infusion, however, after 5-7 days of infusion a progressive return of spasticity was noted, where baseline PMR values returned to preinfusion levels. At the same time, the efficacy of bolus i.t. baclofen treatment also decreased, where after 5 days of baclofen infusion 1.0 microg (i.t.) baclofen only reduced PMR by 10% (compared to 40-50% preinfusion). Baclofen efficacy progressively returned once continuous infusion was stopped. These data demonstrate that transient spinal ischemia leads to the development of spasticity which is sensitive to spinal baclofen. Chronic i.t. infusion leads to a progressive development of tolerance. This model offers potential to study tolerance mechanisms after spinal injury, and aid in drug discovery for use in baclofen-tolerant patients.

Measurement of peripheral muscle resistance in rats with chronic ischemia-induced paraplegia or morphine-induced rigidity using a semi-automated computer-controlled muscle resistance meter.

In experimental and clinical studies, an objective assessment of peripheral muscle resistance represents one of the key elements in determining the efficacy of therapeutic manipulations (e.g. pharmacological, surgical) aimed to ameliorate clinical signs of spasticity and/or rigidity. In the present study, we characterize a newly developed limb flexion resistance meter which permits a semi-automated, computer-controlled measurement of peripheral muscle resistance (PMR) in the lower extremities during a forced flexion of the ankle in the awake rat. Ischemic paraplegia was induced in Sprague-Dawley rats by transient aortic occlusion (10 min) in combination with systemic hypotension (40 mm Hg). After ischemia the presence of spasticity component was determined by the presence of an exaggerated EMG activity recorded from gastrocnemius muscle after nociceptive or proprioceptive afferent activation and by velocity-dependent increase in muscle resistance. Rigidity was induced by high dose (30 mg/kg, i.p.) of morphine. Animals with defined ischemic spasticity or morphine-induced rigidity were then placed into a plastic restrainer and a hind paw attached by a tape to a metal plate driven by a computer-controlled stepping motor equipped with a resistance transducer. The resistance of the ankle to rotation was measured under several testing paradigms: (i) variable degree of ankle flexion (40 degrees, 50 degrees, and 60 degrees), (ii) variable speed/rate of ankle flexion (2, 3, and 4 sec), (iii) the effect of inhalation anesthesia, (iv) the effect of intrathecal baclofen, (v) the effect of dorsal L2-L5 rhizotomy, or (vi) systemic naloxone treatment. In animals with ischemic paraplegia an increased EMG response after peripheral nociceptive or proprioceptive activation was measured. In control animals average muscle resistance was 78 mN and was significantly increased in animals with ischemic spasticity (981-7900 mN). In ischemic-spastic animals a significant increase in measured muscle resistance was seen after increased velocity (4 > 3 > 2 sec) and the angle (40 degrees > 50 degrees > 60 degrees) of the ankle rotation. In spastic animals, deep halothane anesthesia, intrathecal baclofen or dorsal rhizotomy decreased muscle resistance to 39-80% of pretreatment values. Systemic treatment with morphine induced muscle rigidity and corresponding increase in muscle resistance. Morphine-induced increase in muscle resistance was independent on the velocity of the ankle rotation and was reversed by naloxone. These data show that by using this system it is possible to objectively measure the degree of peripheral muscle resistance. The use of this system may represent a simple and effective experimental tool in screening new pharmacological compounds and/or surgical manipulations targeted to modulate spasticity and/or rigidity after a variety of neurological disorders such as spinal cord traumatic or ischemic injury, multiple sclerosis, cerebral palsy, or Parkinson's disease.

Synaptogenesis and amino acid release from long term embryonic rat spinal cord neuronal culture using tissue culture inserts.

In the present study, using tissue culture inserts (TCI) coupled with a primary spinal cord neuronal culture, we characterize a new perfusion system, which permits continuous perfusate collection from cultured neurons. Primary spinal cord neurons were isolated from the lumbar portion of E14 spinal cords of Sprague-Dawley rats, plated on TCI and fed with DMEM/B27/10% FBS. At 1-4 weeks after isolation the development of synapses and neurotransmitter phenotype in cultured neurons was verified using immunofluorescence. A time-dependent development of synapses (Syn) was seen with a dense Syn-positive network identified at 3-4 weeks after plating. A sub-population of plated neurons (35-40%) showed GABA immunoreactivity and expressed NMDAR1 receptor. To measure neurotransmitter release, a chamber accommodating TCI was constructed permitting perfusion of the insert across the membrane. To evoke amino acid release from cultured neurons, NMDA (10 mmol/l) was added into the perfusion buffer. Stimulation with NMDA evoked a significant GABA (4050 +/- 950%) and glutamate release (130 +/- 42%) during first 10 min after exposure. In control non-stimulated cells no significant changes were measured. These data show that by using TCI it is possible to maintain embryonic spinal cord neurons for an extended period and that this system may represent a simple tool to identify neurotransmitter and/or peptides associated with a specific population of cultured brain and/or spinal cord neurons.

Topical bicuculline to the rat spinal cord induces highly localized allodynia that is mediated by spinal prostaglandins.

The purpose of this study was to investigate the allodynic effect of bicuculline (BIC) given topically to the dorsal surface of the rat spinal cord, and to determine if spinal prostaglandins (PGs) mediate the allodynic state arising from spinal GABA(A)-receptor blockade. Male Sprague-Dawley rats (325-400 g) were anaesthetized with halothane and maintained with urethane for the continuous monitoring of blood pressure (MAP), heart rate (HR) and cortical electroencephalogram (EEG). A laminectomy was performed to expose the dorsal surface of the spinal cord. Unilateral application of BIC (0.1 microg in 0.1 microl) to the L5 or L6 spinal segment induced a highly localized allodynia (e.g. one or two digits) on the ipsilateral hind paw. Thus, hair deflection (brushing the hair with a cotton-tipped applicator) in the presence, but not absence of BIC, evoked an increase in MAP and HR, abrupt motor responses (MR; e.g. withdrawal of the hind leg, kicking, and/or scratching) on the affected side, and desynchrony of the EEG. BIC-allodynia was dose-dependent, yielding ED(50)'s (95% CI's) of 45 ng (31-65) for MAP; 68 ng (46-101) for HR and 76 ng (60-97) for MR. Allodynia was sustained for up to 2 h with repeated BIC application without any detectable change in the location or area of peripheral sensitization. Pretreatment with either the EP(1)- receptor antagonist, SC-51322, the cyclooxygenase (COX)-2 selective inhibitor, NS-398, or the NMDA-receptor antagonist, AP-7, inhibited BIC-allodynia in a dose-dependent manner. The results demonstrate: (a) BIC, applied to the dorsal surface of the spinal cord, induces highly localized allodynia; (b) this effect can be sustained with repeated BIC application; (c) it is evoked by NMDA-dependent afferent input; (d) spinal PGs are synthesized by constitutive COX-2 during BIC-allodynia; and (e) spinal PGs contribute to the abnormal processing of tactile input via spinal EP1-receptors.

Spinal nerve injury activates prostaglandin synthesis in the spinal cord that contributes to early maintenance of tactile allodynia.

To determine if spinal prostaglandins (PG) contribute to tactile allodynia, male, Sprague-Dawley rats were fitted with either intrathecal (i.t.) microdialysis or drug delivery catheters 3 days before tight ligation of the left lumber 5/6 spinal nerves. Paw withdrawal thresholds (PWT) were determined using von Frey filaments. Ligated rats developed tactile allodynia within 24h, as evidenced by a decrease in PWT in the affected hindpaw (<4 g vs. >15 g control). Sham-operated controls were unchanged from baseline (>15 g). Allodynia was also characterized by a significant increase in the evoked release of PGE(2). Thus, brushing the plantar surface of the affected hindpaw with a cotton-tipped applicator, 5 days postligation, increased the [PGE(2)](dialysate) to 199+/-34% of the prestimulus control period. In contrast, brushing had no detectable effect on release before surgery or in sham-operated animals. Basal release (no brushing) was similar before and after surgery (sham-operated and ligated rats). In a separate group of rats and beginning 2 days after ligation, the acute i.t. injection of S(+)-ibuprofen, SC-51322, SC-236, or SC-560 significantly reversed allodynia (maximum effect=69+/-9, 66+/-6, 57+/-4, 20+/-5%, respectively). R(-)-ibuprofen or vehicle were without effect. The results of this study suggest that: (a). spinal PG synthesis and allodynia-like behaviour are triggered by normally innocuous brushing after spinal nerve ligation; (b). pharmacological disruption of this cascade significantly reverses allodynia; (c). COX-2 is the relevant isozyme; and (d). the PG effect is mediated by spinal EP receptors.

Differential distribution of PI3K isoforms in spinal cord and dorsal root ganglia: potential roles in acute inflammatory pain.

PI3-kinases (PI3Ks) participate in nociception within spinal cord, dorsal root ganglion (DRG), and peripheral nerves. To extend our knowledge, we immunohistochemically stained for each of the 4 class I PI3K isoforms along with several cell-specific markers within the lumbar spinal cord, DRG, and sciatic nerve of naive rats. Intrathecal and intraplantar isoform specific antagonists were given as pretreatments before intraplantar carrageenan; pain behavior was then assessed over time. The α-isoform was localized to central terminals of primary afferent fibers in spinal cord laminae IIi to IV as well as to neurons in ventral horn and DRG. The PI3Kß isoform was the only class I isoform seen in dorsal horn neurons; it was also observed in DRG, Schwann cells, and axonal paranodes. The δ-isoform was found in spinal cord white matter oligodendrocytes and radial astrocytes, and the γ-isoform was seen in a subpopulation of IB4-positive DRG neurons. No isoform co-localized with microglial markers or satellite cells in naive tissue. Only the PI3Kß antagonist, but none of the other antagonists, had anti-allodynic effects when administered intrathecally; coincident with reduced pain behavior, this agent completely blocked paw carrageenan-induced dorsal horn 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic acid (AMPA) receptor trafficking to plasma membranes. Intraplantar administration of the γ-antagonist prominently reduced pain behavior. These data suggest that each isoform displays specificity with regard to neuronal type as well as to specific tissues. Furthermore, each PI3K isoform has a unique role in development of nociception and tissue inflammation.

Pig models of neurodegenerative disorders: Utilization in cell replacement-based preclinical safety and efficacy studies.

An important component for successful translation of cell replacement-based therapies into clinical practice is the utilization of large animal models to conduct efficacy and/or safety cell dosing studies. Over the past few decades, several large animal models (dog, cat, nonhuman primate) were developed and employed in cell replacement studies; however, none of these models appears to provide a readily available platform to conduct effective and large-scale preclinical studies. In recent years, numerous pig models of neurodegenerative disorders were developed using both a transgenic approach as well as invasive surgical techniques. The pig model (naïve noninjured animals) was recently used successfully to define the safety and optimal dosing of human spinal stem cells after grafting into the central nervous system (CNS) in immunosuppressed animals. The data from these studies were used in the design of a human clinical protocol used in amyotrophic lateral sclerosis (ALS) patients in a Phase I clinical trial. In addition, a highly inbred (complete major histocompatibility complex [MHC] match) strain of miniature pigs is available which permits the design of comparable MHC combinations between the donor cells and the graft recipient as used in human patients. Jointly, these studies show that the pig model can represent an effective large animal model to be used in preclinical cell replacement modeling. This review summarizes the available pig models of neurodegenerative disorders and the use of some of these models in cell replacement studies. The challenges and potential future directions in more effective use of the pig neurodegenerative models are also discussed.

Effective long-term immunosuppression in rats by subcutaneously implanted sustained-release tacrolimus pellet: effect on spinally grafted human neural precursor survival.

Achievement of effective, safe and long-term immunosuppression represents one of the challenges in experimental allogeneic and xenogeneic cell and organ transplantation. The goal of the present study was to develop a reliable, long-term immunosuppression protocol in Sprague-Dawley (SD) rats by: 1) comparing the pharmacokinetics of four different subcutaneously delivered/implanted tacrolimus (TAC) formulations, including: i) caster oil/saline solution, ii) unilamellar or multilamellar liposomes, iii) biodegradable microspheres, and iv) biodegradable 3-month lasting pellets; and 2) defining the survival and immune response in animals receiving spinal injections of human neural precursors at 6 weeks to 3 months after cell grafting. In animals implanted with TAC pellets (3.4 mg/kg/day), a stable 3-month lasting plasma concentration of TAC averaging 19.1 ± 4.9 ng/ml was measured. Analysis of grafted cell survival in SOD+ or spinal trauma-injured SD rats immunosuppressed with 3-month lasting TAC pellets (3.4-5.1 mg/kg/day) showed the consistent presence of implanted human neurons with minimal or no local T-cell infiltration. These data demonstrate that the use of TAC pellets can represent an effective, long-lasting immunosuppressive drug delivery system that is safe, simple to implement and is associated with a long-term human neural precursor survival after grafting into the spinal cord of SOD+ or spinal trauma-injured SD rats.

Human neural stem cell replacement therapy for amyotrophic lateral sclerosis by spinal transplantation.

BACKGROUND: Mutation in the ubiquitously expressed cytoplasmic superoxide dismutase (SOD1) causes an inherited form of Amyotrophic Lateral Sclerosis (ALS). Mutant synthesis in motor neurons drives disease onset and early disease progression. Previous experimental studies have shown that spinal grafting of human fetal spinal neural stem cells (hNSCs) into the lumbar spinal cord of SOD1(G93A) rats leads to a moderate therapeutical effect as evidenced by local α-motoneuron sparing and extension of lifespan. The aim of the present study was to analyze the degree of therapeutical effect of hNSCs once grafted into the lumbar spinal ventral horn in presymptomatic immunosuppressed SOD1(G93A) rats and to assess the presence and functional integrity of the descending motor system in symptomatic SOD1(G93A) animals. METHODS/PRINCIPAL FINDINGS: Presymptomatic SOD1(G93A) rats (60-65 days old) received spinal lumbar injections of hNSCs. After cell grafting, disease onset, disease progression and lifespan were analyzed. In separate symptomatic SOD1(G93A) rats, the presence and functional conductivity of descending motor tracts (corticospinal and rubrospinal) was analyzed by spinal surface recording electrodes after electrical stimulation of the motor cortex. Silver impregnation of lumbar spinal cord sections and descending motor axon counting in plastic spinal cord sections were used to validate morphologically the integrity of descending motor tracts. Grafting of hNSCs into the lumbar spinal cord of SOD1(G93A) rats protected α-motoneurons in the vicinity of grafted cells, provided transient functional improvement, but offered no protection to α-motoneuron pools distant from grafted lumbar segments. Analysis of motor-evoked potentials recorded from the thoracic spinal cord of symptomatic SOD1(G93A) rats showed a near complete loss of descending motor tract conduction, corresponding to a significant (50-65%) loss of large caliber descending motor axons. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that in order to achieve a more clinically-adequate treatment, cell-replacement/gene therapy strategies will likely require both spinal and supraspinal targets.

Combinational spinal GAD65 gene delivery and systemic GABA-mimetic treatment for modulation of spasticity.

BACKGROUND: Loss of GABA-mediated pre-synaptic inhibition after spinal injury plays a key role in the progressive increase in spinal reflexes and the appearance of spasticity. Clinical studies show that the use of baclofen (GABA(B) receptor agonist), while effective in modulating spasticity is associated with major side effects such as general sedation and progressive tolerance development. The goal of the present study was to assess if a combined therapy composed of spinal segment-specific upregulation of GAD65 (glutamate decarboxylase) gene once combined with systemic treatment with tiagabine (GABA uptake inhibitor) will lead to an antispasticity effect and whether such an effect will only be present in GAD65 gene over-expressing spinal segments. METHODS/PRINCIPAL FINDINGS: Adult Sprague-Dawley (SD) rats were exposed to transient spinal ischemia (10 min) to induce muscle spasticity. Animals then received lumbar injection of HIV1-CMV-GAD65 lentivirus (LVs) targeting ventral α-motoneuronal pools. At 2-3 weeks after lentivirus delivery animals were treated systemically with tiagabine (4, 10, 20 or 40 mg/kg or vehicle) and the degree of spasticity response measured. In a separate experiment the expression of GAD65 gene after spinal parenchymal delivery of GAD65-lentivirus in naive minipigs was studied. Spastic SD rats receiving spinal injections of the GAD65 gene and treated with systemic tiagabine showed potent and tiagabine-dose-dependent alleviation of spasticity. Neither treatment alone (i.e., GAD65-LVs injection only or tiagabine treatment only) had any significant antispasticity effect nor had any detectable side effect. Measured antispasticity effect correlated with increase in spinal parenchymal GABA synthesis and was restricted to spinal segments overexpressing GAD65 gene. CONCLUSIONS/SIGNIFICANCE: These data show that treatment with orally bioavailable GABA-mimetic drugs if combined with spinal-segment-specific GAD65 gene overexpression can represent a novel and highly effective anti-spasticity treatment which is associated with minimal side effects and is restricted to GAD65-gene over-expressing spinal segments.

Optimization of immunosuppressive therapy for spinal grafting of human spinal stem cells in a rat model of ALS.

Previous rodent studies employing monotherapy or combined immunosuppressive regimens have demonstrated a variable degree of spinal xenograft survival in several spinal neurodegenerative models including spinal ischemia, trauma, or amyotrophic lateral sclerosis (ALS). Accordingly, the characterization of optimal immunosuppressive protocols for the specific neurodegenerative model is critical to ensure reliable assessment of potential long-term therapeutic effects associated with cell replacement. In the present study we characterized the survival of human spinal stem cells when grafted into the lumbar spinal cords of a rodent model of ALS, SOD1 (G93A) male and female rats (60-67 days old). Four different immunosuppressive protocols were studied: i) FK506 (q12h); ii) FK506 (qd) + mycophenolate (PO; q12h, up to 7 days postop); iii) FK506 (qd) + mycophenolate (IP; q12h, up to 7 days postop); and iv) FK506 (qd) + mycophenolate (IP; qd, up to 7 days postop). Three weeks after cell grafting the number of surviving human cells was then systematically assessed. The highest density of grafted cells was seen in animals treated with FK506 (qd) and mycophenolate (IP; qd; an average 915 ± 95 grafted cells per spinal cord section). The majority of hNUMA-positive cells colocalized with doublecortin (DCX) immunoreactivity. DCX-positive neurons showed extensive axodendritic sprouting toward surrounding host neurons. In addition, migrating grafted cells were identified up to 500 µm from the graft. In animals treated with FK506 (q12h), FK506 (qd) + mycophenolate (PO; q12h) or FK506 (qd) + mycophenolate (IP; q12h), 11.8 ± 3.4%, 61.2 ± 7.8%, and 99.4 ± 8.9% [expressed as percent of the FK506 (qd) and mycophenolate (IP; qd)] cell survival was seen, respectively. In contrast to animals treated with a combination of FK506 + mycophenolate, robust CD4/8 immunoreactivity was identified in the vicinity of the injection tract in animals treated with FK506 only. These data suggest that a combined, systemically delivered immunosuppression regimen including FK506 and mycophenolate can significantly improve survival of human spinal stem cells after intraspinal transplantation in SOD1 (G93A) rats.

Suppression of stretch reflex activity after spinal or systemic treatment with AMPA receptor antagonist NGX424 in rats with developed baclofen tolerance.

BACKGROUND AND PURPOSE: Baclofen (a GABA(B) receptor agonist) is the most commonly used anti-spasticity agent in clinical practice. While effective when administered spinally or systemically, the development of progressive tolerance represents a serious limitation for its long-term use. The goal of the present study was to characterize the treatment potency after intrathecal or systemic treatment with the selective AMPA receptor antagonist NGX424 on stretch reflex activity (SRA) and background muscle activity (BMA) in rats with developed baclofen tolerance. EXPERIMENTAL APPROACH: Animals were exposed to 10 min of spinal ischaemia to induce an increase in BMA and SRA. Selected animals were implanted with an intrathecal PE-5 catheter and infused intrathecally with baclofen (1 µg·h⁻¹ ) for 14 days. Before and after baclofen infusion, changes in BMA and SRA were measured at 2 day intervals. After development of baclofen tolerance, the animals were injected intrathecally (1 µg) or subcutaneously (3, 6 or 12 mg·kg⁻¹) with NGX424, and changes in BMA and SRA were measured. KEY RESULTS: Intrathecal or systemic delivery of NGX424 significantly suppressed the BMA and SRA in baclofen-tolerant animals. This effect was dose dependent. The magnitude of BMA and SRA suppression seen after 1 µg (intrathecal) or 12 mg·kg ⁻¹ (s.c.) of NGX424 injection was similar to that seen during the first 5 days of baclofen infusion. CONCLUSIONS AND IMPLICATIONS These data demonstrate that the use of NGX424 can represent an effective therapy to modulate chronic spasticity in patients who are refractory or tolerant to baclofen treatment.