SOD1 Rats Research Articles

Does oestradiol treatment alleviate obesity-induced oxidative stress in the male liver?

The liver is an organ in which many oxidative processes occur and represents an important target of oxidative stress (OxS). Under physiological conditions of normal mitochondrial homeostasis, hepatocytes effectively remove reactive oxygen species (ROS) by enabling metabolic adaptations and through the antioxidant defence system mechanisms. However, obesity-induced lipid accumulation in the hepatocytes causes significantly elevated production of ROS, reduces oxidative capacity, and increases oxidative stress (OxS). In men, compared with premenopausal women, the development of insulin resistance (IR) and non-alcoholic fatty liver disease (NAFLD) in obesity are more prevalent, where oestradiol (E2), the most potent female sex steroid, is proposed as the main culprit. Exogenous oestradiol (E2) administration exerts beneficial effects on antioxidant properties, restores total plasma antioxidant capacity and decreases biomarkers of OxS in ovariectomized animal models. Thus, we hypothesized that E2 treatment in states of obesity could have beneficial effects againstOxS in the obese male's liver. We assumed that E2 could directly affect the level of OxS by increasing the level/activity of the AOS enzymes, particularly SOD1, SOD2, GPx, and CAT, in obese males' livers. In addition, we assumed that the level of malondialdehyde (MDA) and protein carbonyl content (PCC) in obese males' livers would be reduced after E2 treatment as a result of E2 inhibitory effect on lipid peroxidation and protein oxidation, respectively. To test our hypothesis, we used the liver of a high-fat (HF) diet-fed male Wistar rats as an animal model of obesity, treated with E2 intraperitoneally (40 μg/kg). Preliminary results from this study support our hypothesis that E2 increases liver protein expression of AOS enzymes: SOD1, GPx, and CAT, in control and HF male rats compared with their respective controls. The protein level of SOD2 and CAT activity was increased in HF treated with E2 compared with non-treated HF rats. Moreover, as we expected, E2 administration significantly decreased the MDA level in both E2-treated groups compared to their controls, while the PCC level was significantly decreased in HF treated group compared with untreated HF rats. In conclusion, the preliminary results we obtained in this study indicate that E2 administration can effectively inhibit the OxS-related processes in the liver in HF diet-induced obesity by increasing AOS enzymes levels and CAT activity, and also by decreasing levels of MDA and PCC. A consequence of our hypothesis is that treatment with E2 may be an innovative way to improve obesity-related liver disease prevention and healing.

Transplantation of clinical-grade human neural stem cells reduces neuroinflammation, prolongs survival and delays disease progression in the SOD1 rats

Stem cells are emerging as a therapeutic option for incurable diseases, such as Amyotrophic Lateral Sclerosis (ALS). However, critical issues are related to their origin as well as to the need to deepen our knowledge of the therapeutic actions exerted by these cells. Here, we investigate the therapeutic potential of clinical-grade human neural stem cells (hNSCs) that have been successfully used in a recently concluded phase I clinical trial for ALS patients (NCT01640067). The hNSCs were transplanted bilaterally into the anterior horns of the lumbar spinal cord (four grafts each, segments L3–L4) of superoxide dismutase 1 G93A transgenic rats (SOD1 rats) at the symptomatic stage. Controls included untreated SOD1 rats (CTRL) and those treated with HBSS (HBSS). Motor symptoms and histological hallmarks of the disease were evaluated at three progressive time points: 15 and 40 days after transplant (DAT), and end stage. Animals were treated by transient immunosuppression (for 15 days, starting at time of transplantation). Under these conditions, hNSCs integrated extensively within the cord, differentiated into neural phenotypes and migrated rostro-caudally, up to 3.77 ± 0.63 cm from the injection site. The transplanted cells delayed decreases in body weight and deterioration of motor performance in the SOD1 rats. At 40DAT, the anterior horns at L3–L4 revealed a higher density of motoneurons and fewer activated astroglial and microglial cells. Accordingly, the overall survival of transplanted rats was significantly enhanced with no rejection of hNSCs observed. We demonstrated that the beneficial effects observed after stem cell transplantation arises from multiple events that counteract several aspects of the disease, a crucial feature for multifactorial diseases, such as ALS. The combination of therapeutic approaches that target different pathogenic mechanisms of the disorder, including pharmacology, molecular therapy and cell transplantation, will increase the chances of a clinically successful therapy for ALS.

A Combination of Intrathecal and Intramuscular Application of Human Mesenchymal Stem Cells Partly Reduces the Activation of Necroptosis in the Spinal Cord of SOD1G93A Rats

An increasing number of studies have demonstrated the beneficial effects of human mesenchymal stem cells (hMSC) in the treatment of amyotrophic lateral sclerosis (ALS). We compared the effect of repeated intrathecal applications of hMSC or their conditioned medium (CondM) using lumbar puncture or injection into the muscle (quadriceps femoris), or a combination of both applications in symptomatic SOD1G93A rats. We further assessed the effect of the treatment on three major cell death pathways (necroptosis, apoptosis, and autophagy) in the spinal cord tissue. All the animals were behaviorally tested (grip strength test, Basso Beattie Bresnahan (BBB) test, and rotarod), and the tissue was analyzed immunohistochemically, by qPCR and Western blot. All symptomatic SOD1 rats treated with hMSC had a significantly increased lifespan, improved motor activity and reduced number of Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive cells. Moreover, a combined hMSC delivery increased motor neuron survival, maintained neuromuscular junctions in quadriceps femoris and substantially reduced the levels of proteins involved in necroptosis (Rip1, mixed lineage kinase‐like protein, cl‐casp8), apoptosis (cl‐casp 9) and autophagy (beclin 1). Furthermore, astrogliosis and elevated levels of Connexin 43 were decreased after combined hMSC treatment. The repeated application of CondM, or intramuscular injections alone, improved motor activity; however, this improvement was not supported by changes at the molecular level. Our results provide new evidence that a combination of repeated intrathecal and intramuscular hMSC applications protects motor neurons and neuromuscular junctions, not only through a reduction of apoptosis and autophagy but also through the necroptosis pathway, which is significantly involved in cell death in rodent SOD1G93A model of ALS. stem cells translational medicine 2019;8:535–547

Mutant SOD1 prevents normal functional recovery through enhanced glial activation and loss of motor neuron innervation after peripheral nerve injury

BackgroundAmyotrophic lateral sclerosis (ALS) is poorly understood with no effective therapeutics. One long entertained observation is that ALS may be precipitated focally by nerve injury. Many patients with ALS are athletes or veterans, and some have suffered nerve injuries at the site where ALS first presents. Here we explore how a genetic SOD1 mutation alters the inflammatory response and affects functional recovery after an environmental insult in a rat model. MethodsUnilateral sciatic nerve crush injuries were performed in SOD1 G93A rats prior to disease symptom onset. Functional recovery was compared between injured wild-type littermates and uninjured SOD1 rats. Spinal cord tissues were analyzed quantitatively for SOD1 expression, glial reactivity, and motor neuron synaptic integrity. ResultsInjured SOD1 rats failed to recover and showed hastened functional decline with decreased survival. Injury induced extracellular SOD1 expression was associated with heightened, prolonged microglial and astrogial activation in the ventral horn. This inflammatory response spread to uninjured motor neuron pools and was associated with increased motor neuron synaptic loss. DiscussionThis study identified a relationship between genetic and environmental contributions to disease onset and progression in ALS. The findings suggest that injury induced SOD1 mutant protein induces a heightened and prolonged inflammatory response resulting in motor neuron degeneration through synaptic loss. Once initiated, this process spreads to adjacent motor neurons leading to contiguous spread of the disease. Treatments that suppress this heightened glial response could slow disease progression in ALS patients with focal sites of disease onset. Significance statementThe contribution of environmental factors such as peripheral nerve insults in ALS is not well understood. Here we examined the effect of a single sciatic nerve injury in SOD1 (G93A) rats to explore the contribution of this environmental insult on disease onset and progression. After the injury, SOD1 animals failed to recover and had a more rapid functional decline. Histopathologically, SOD1 animals had heightened SOD1 expression, microglial and astroglial responses, and a reduction of motor neuron innervation. Taken together, these results provide a plausible mechanism of how the SOD1 mutated protein promotes an abnormal response to injury that leads to neurodegenerative changes in an ALS model that is amenable to therapeutic testing.

A model of recurrent concussion that leads to long-term motor deficits, CTE-like tauopathy and exacerbation of an ALS phenotype.

Concussion injury is the most common form of traumatic brain injury (TBI). How recurrent concussions alter long-term outcomes is poorly understood, especially as related to the development of neurodegenerative disease. We evaluated the functional and pathological consequences of repeated TBI over time in wild type (WT) rats as well as rats harboring the human SOD1 mutation ("SOD1"), a model of familial amyotrophic lateral sclerosis (ALS). A total of 42 rats, 26 WT and 16 SOD1, were examined over a study period of 25 weeks (or endpoint). At postnatal day 60, 20 WT and 7 SOD1 rats were exposed to mild, bilateral TBI once per week for either 2 weeks (2×TBI) or 5 weeks (5×TBI) using a controlled cortical impact device. Six WT and nine SOD1 rats underwent sham injury with anesthesia alone. Twenty WT rats were euthanized at 12 weeks after first injury and six WT rats were euthanized at 25 weeks after first injury. SOD1 rats were euthanized when they reached ALS disease endpoint. Weekly body weights and behavioral assessments were performed. Tauopathy in brain tissue was analyzed using immunohistochemistry. 2XTBI injured rats initially demonstrated recovery of motor function but failed to recover to baseline within the 12-week study period. Relative to both 2XTBI and sham controls, 5XTBI rats demonstrated significant deficits that persisted over the 12-week period. SOD1 5XTBI rats reached a peak body weight earlier than sham SOD1 rats, indicating earlier onset of the ALS phenotype. Histologic examination of brain tissue revealed that, in contrast with sham controls, SOD1 and WT TBI rats demonstrated cortical and corpus collosum thinning and tauopathy, which increased over time. Unlike previous models of repeat brain injury, which demonstrate only transient deficits in motor function, our concussion model of repeat, mild, bilateral TBI induced long-lasting deficits in motor function, decreased cortical thickness, shrinkage of the corpus callosum, increased brain tauopathy, and earlier onset of ALS symptoms in SOD1 rats. This model may allow for a greater understanding of the complex relationship between TBI and neurodegenerative diseases and provides a potential method for testing novel therapeutic strategies.

Post-paralysis tyrosine kinase inhibition with masitinib abrogates neuroinflammation and slows disease progression in inherited amyotrophic lateral sclerosis.

BackgroundIn the SOD1G93A mutant rat model of amyotrophic lateral sclerosis (ALS), neuronal death and rapid paralysis progression are associated with the emergence of activated aberrant glial cells that proliferate in the degenerating spinal cord. Whether pharmacological downregulation of such aberrant glial cells will decrease motor neuron death and prolong survival is unknown. We hypothesized that proliferation of aberrant glial cells is dependent on kinase receptor activation, and therefore, the tyrosine kinase inhibitor masitinib (AB1010) could potentially control neuroinflammation in the rat model of ALS.MethodsThe cellular effects of pharmacological inhibition of tyrosine kinases with masitinib were analyzed in cell cultures of microglia isolated from aged symptomatic SOD1G93A rats. To determine whether masitinib prevented the appearance of aberrant glial cells or modified post-paralysis survival, the drug was orally administered at 30 mg/kg/day starting after paralysis onset.ResultsWe found that masitinib selectively inhibited the tyrosine kinase receptor colony-stimulating factor 1R (CSF-1R) at nanomolar concentrations. In microglia cultures from symptomatic SOD1G93A spinal cords, masitinib prevented CSF-induced proliferation, cell migration, and the expression of inflammatory mediators. Oral administration of masitinib to SOD1G93A rats starting after paralysis onset decreased the number of aberrant glial cells, microgliosis, and motor neuron pathology in the degenerating spinal cord, relative to vehicle-treated rats. Masitinib treatment initiated 7 days after paralysis onset prolonged post-paralysis survival by 40 %.ConclusionsThese data show that masitinib is capable of controlling microgliosis and the emergence/expansion of aberrant glial cells, thus providing a strong biological rationale for its use to control neuroinflammation in ALS. Remarkably, masitinib significantly prolonged survival when delivered after paralysis onset, an unprecedented effect in preclinical models of ALS, and therefore appears well-suited for treating ALS.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0620-9) contains supplementary material, which is available to authorized users.

Abstract W P358: SOD overexpression Accounts for the Neurovascular Protection in Rats Following Ischemic Stroke

Acute hyperglycemic stroke (AHS) is associated with poor stroke outcomes and functional recovery. We recently showed that myogenic response; the ability of blood vessels to maintain adequate blood flow despite changes in pressure; was impaired after AHS. Experimental studies showed that acute elevation of blood glucose at stroke onset enhances oxidative stress that impairs effective reperfusion. Copper/zinc-superoxide dismutase (SOD1), an antioxidant enzyme that catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide, has been shown to be highly protective against ischemia/reperfusion injury (IRI). Yet, the mechanism by which SOD1 improves stroke outcomes is still unresolved. Hypothesis: SOD1 overexpression improves cerebral myogenic behavior leading to neurovascular protection after AHS Methods: Wild-type Sprague Dawley (SD) and human SOD1 transgenic rats (n=6-8) were subjected to 30 min middle cerebral artery occlusion (MCAO)/45 min or 24 hr reperfusion. Hyperglycemia was induced by intraperitoneal injection of 40 % glucose solution 10 min before procedure. Myogenic tone of isolated MCAs, infarct size, edema and neurological deficits were determined. Results: IRI impaired myogenic response of MCAs isolated from SD rats compared to sham (***p<0.001). Normoglycemic and hyperglycemic SOD1 rats maintained a well developed myogenic response after IRI. Infarct size and edema were significantly reduced in the SOD1 transgenic rats compared to SD rats (**p<0.01). SOD1 overexpression significantly improved beam walk score and grip strength deficit compared to SD rats (*p<0.05). Hyperglycemic SOD1 transgenic rats showed smaller infarct size and edema (**p<0.01) and experienced improved neurological behavior (*p<0.05) compared to hyperglycemic SD rats. Conclusions: We provide evidence that SOD1 overexpression preserves the myogenic behavior of MCAs achieving neurovascular protection in conditions associated with poor stroke outcomes. Further studies are required to unravel the exact mechanism by which SOD exerts its beneficial role on vascular reactivity, and to identify new therapeutic targets for stroke management.

Progranulin does not affect motor neuron degeneration in mutant SOD1 mice and rats

Motor neuron degeneration in amyotrophic lateral sclerosis (ALS) is familial in 10% of patients, with mutations in SOD1 and C9orf72 being the most frequent cause. There is convincing evidence for overlap between ALS and frontotemporal lobar degeneration at the genetic, pathological, and clinical level. Null mutations in progranulin (PGRN) are a frequent cause of familial frontotemporal lobar degeneration. PGRN exerts neurotrophic properties on motor neurons in vitro and in vivo. We therefore examined whether PGRN could affect disease progression in mutant SOD1 mice and rats, both established models for ALS. Overexpression of PGRN in mice and intracerebroventricular delivery of PGRN in rats did not affect onset or progression of motor neuron degeneration.

Neural Progenitors Derived From Human Induced Pluripotent Stem Cells Survive and Differentiate Upon Transplantation Into a Rat Model of Amyotrophic Lateral Sclerosis

Human induced pluripotent stem cells (iPSCs) offer hope for personalized regenerative cell therapy in amyotrophic lateral sclerosis (ALS). We analyzed the fate of human iPSC-derived neural progenitors transplanted into the spinal cord of wild-type and transgenic rats carrying a human mutated SOD1(G93A) gene. The aim was to follow survival and differentiation of human neural progenitors until day 60 post-transplantation in two different in vivo environments, one being ALS-like. iPSC-derived neural progenitors efficiently engrafted in the adult spinal cord and survived at high numbers. Different neural progenitor, astroglial, and neuronal markers indicated that, over time, the transplanted nestin-positive cells differentiated into cells displaying a neuronal phenotype in both wild-type and transgenic SOD1 rats. Although a transient microglial phenotype was detected at day 15, astroglial staining was negative in engrafted cells from day 1 to day 60. At day 30, differentiation toward a neuronal phenotype was identified, which was further established at day 60 by the expression of the neuronal marker MAP2. A specification process into motoneuron-like structures was evidenced in the ventral horns in both wild-type and SOD1 rats. Our results demonstrate proof-of-principle of survival and differentiation of human iPSC-derived neural progenitors in in vivo ALS environment, offering perspectives for the use of iPSC-based therapy in ALS.

Phenotypic transition of microglia into astrocyte-like cells associated with disease onset in a model of inherited ALS

Microglia and reactive astrocytes accumulate in the spinal cord of rats expressing the Amyotrophic lateral sclerosis (ALS)-linked SOD1 G93A mutation. We previously reported that the rapid progression of paralysis in ALS rats is associated with the appearance of proliferative astrocyte-like cells that surround motor neurons. These cells, designated as Aberrant Astrocytes (AbA cells) because of their atypical astrocytic phenotype, exhibit high toxicity to motor neurons. However, the cellular origin of AbA cells remains unknown. Because AbA cells are labeled with the proliferation marker Ki67, we analyzed the phenotypic makers of proliferating glial cells that surround motor neurons by immunohistochemistry. The number of Ki67 +AbA cells sharply increased in symptomatic rats, displaying large cell bodies with processes embracing motor neurons. Most were co-labeled with astrocytic marker GFAP concurrently with the microglial markers Iba1 and CD163. Cultures of spinal cord prepared from symptomatic SOD1 G93A rats yielded large numbers of microglia expressing Iba1, CD11b, and CD68. Cells sorted for CD11b expression by flow cytometry transformed into AbA cells within two weeks. During these two weeks, the expression of microglial markers largely disappeared, while GFAP and S100β expression increased. The phenotypic transition to AbA cells was stimulated by forskolin. These findings provide evidence for a subpopulation of proliferating microglial cells in SOD1 G93A rats that undergo a phenotypic transition into AbA cells after onset of paralysis that may promote the fulminant disease progression. These cells could be a therapeutic target for slowing paralysis progression in ALS.

Amyotrophic lateral sclerosis: a dying motor unit?

ALS is a late-onset neurodegenerative disease of unknown etiology. Since it was first described (Charcot, 1874), several mechanisms, such as oxidative stress, glutamate excitotoxicity, glial cell pathology, or aberrant RNA metabolism (for review, see Vucic and Kiernan, 2009). Recently the evidence accumulates that mitochondrial damage and defective axonal transport may play a pivotal role (Shi et al., 2010; Cozzolino and Carri, 2012). Nowadays, a multifactorial origin is widely accepted for the neurodegeneration in ALS, but the triggering mechanism(s) underlying its initiation remain to be defined. One of the issues, which are still unresolved, is the triggering site. ALS is classically thought to be a disease that causes the progressive loss of upper and lower motoneurones (MNs) followed by axonal degeneration and muscle atrophy, resulting in the virtually complete disappearance of spinal and cortical MNs (Charcot, 1874; Strong and Rosenfeld, 2003). This point of view is still prevalent among clinical neurologists, and ongoing debates focus mostly on the question of which MN subsystem (upper or lower) is affected first (Chou and Norris, 1993; Eisen et al., 1996; Eisen, 2009; Van Der Graaff et al., 2009). For obvious reasons, it is not possible to directly characterize the properties of MNs in ALS patients. Therefore, a significant proportion of the research on ALS takes place in animal models of the disease that carry mutations in the Cu/Zn superoxide dismutase gene (the SOD1 mice and rats). Although these mutations are responsible only for about 2.5% of all the ALS cases, the pathology in SOD1 animals largely resembles clinical features of human sporadic ALS. In recent years, research in SOD1 animal ALS models has found MN pathology to begin at the distal axon terminals and to proceed in a “dying back” pattern (Fischer et al., 2004; Xie et al., 2005; Parkhouse et al., 2008; Shi et al., 2009; Carrasco et al., 2010). Below, we will present a piece of experimental evidence that, in our opinion, may be interpreted in favor of this last view.

Intermittent Hypoxia and Stem Cell Implants Preserve Breathing Capacity in a Rodent Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease causing paralysis and death from respiratory failure. Strategies to preserve and/or restore respiratory function are critical for successful treatment. Although breathing capacity is maintained until late in disease progression in rodent models of familial ALS (SOD1(G93A) rats and mice), reduced numbers of phrenic motor neurons and decreased phrenic nerve activity are observed. Decreased phrenic motor output suggests imminent respiratory failure. To preserve or restore phrenic nerve activity in SOD1(G93A) rats at disease end stage. SOD1(G93A) rats were injected with human neural progenitor cells (hNPCs) bracketing the phrenic motor nucleus before disease onset, or exposed to acute intermittent hypoxia (AIH) at disease end stage. The capacity to generate phrenic motor output in anesthetized rats at disease end stage was: (1) transiently restored by a single presentation of AIH; and (2) preserved ipsilateral to hNPC transplants made before disease onset. hNPC transplants improved ipsilateral phrenic motor neuron survival. AIH-induced respiratory plasticity and stem cell therapy have complementary translational potential to treat breathing deficits in patients with ALS.

Human Neural Stem Cell Replacement Therapy for Amyotrophic Lateral Sclerosis by Spinal Transplantation

BackgroundMutation 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 SOD1G93A 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 SOD1G93A rats and to assess the presence and functional integrity of the descending motor system in symptomatic SOD1G93A animals.Methods/Principal FindingsPresymptomatic SOD1G93A 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 SOD1G93A 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 SOD1G93A 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 SOD1G93A 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/SignificanceThese 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.

Transplantation and follow-up of human iPSc-derived neural progenitors in a rat model of ALS

Transplantation and follow-up of human iPSc-derived neural progenitors in a rat model of ALS

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.

Motoneuron afterhyperpolarisation duration in amyotrophic lateral sclerosis

Motor unit (MU) potentials were registered from 20 ALS patients and 13 age-matched control individuals during isometric constant force contractions of brachial biceps (BB). The registered signals were decomposed into single MU potential trains. The estimates of duration of the afterhyperpolarisation (AHP) in MNs, derived from the interspike interval variability, was compared between ALS patients (124 MNs) and control subjects (111 MNs) and no significant differences were encountered. However, the relationship between TI and age for patients appeared to be qualitatively different from that of the control group. The dependence of patients' AHPs on relative force deficit (RFD), which quantified muscle involvement, was more specific. For RFDs below 30%, the AHP estimate was significantly lower than control values and then increased thereafter with increasing RFDs. Moreover, firing rates of patients with the smallest RFDs were significantly higher while firing rates of patients with the greatest RFDs were significantly lower than control values. The AHP shortening in the early stages of muscle impairment is consistent with the decrease in firing threshold of ‘fast' MNs found in spinal cord slices from neonatal SOD1 mice. The later elongation of the AHP may be caused by the higher vulnerability of ‘fast' MNs to degeneration and by the influence of reinnervation. Our results are comparable to what has been observed in acute experiments in animal models, providing a bridge between animal and clinical research that may be relevant for identification of mechanism(s) underlying neurodegeneration in ALS.

Olfactory Ensheathing Cell Transplantation Into Spinal Cord Prolongs the Survival of Mutant SOD1G93A ALS Rats Through Neuroprotection and Remyelination

Amyotrophic lateral sclerosis (ALS) is a progressively fatal, incurable, neurodegenerative disorder. In this study, we investigated whether olfactory ensheathing cells (OEC) transplantation could provide protection to motor neurons and enable remyelination in mutant SOD1(G93A) transgenic rats with ALS. Seventy-two rats were divided into four groups: SOD1(G93A) rats (n = 20); medium+SOD1(G93A) rats (n = 20); OECs+SOD1(G93A) rats (n = 24); and another eight wild-type rats were used as controls. About 5 μL (1 × 10(5)) OECs in DF12 medium was injected into the dorsal funiculus of the thoracic spinal cord at a predetermined depth. Survival analysis revealed a significant increase in the survival time in OEC+SOD1(G93A) rats. Body weight records and inclined board test showed a significant difference between OEC+SOD1(G93A) and SOD1(G93A) from the onset at 7 days to 11 days (P < 0.05). Four weeks following transplantation, motor neuron counts in the ventral horn of the spinal cord noted a significant motor neuron loss in SOD1(G93A) rats when compared with wild-type rats (P < 0.001), and much less neuronal loss and collapse was noted in OEC+SOD1(G93A) rats when compared with SOD1(G93A) rats(P < 0.001); immunohistochemistry and Western blot analysis of choline acetyltransferase supported the motor neuron count. Images of confocal microscope indicated that the transplanted OECs had survived for more than 4 weeks and migrated 4.2 mm through the spinal cord. Evidence of remyelination of transplanted OEC was captured with triple fluorescence labeling of green fluorescent protein, neurofilament, and myelin basic protein and was further confirmed by Western blot analysis of MPB. In conclusion, the transplanted OECs could serve as a source of neuroprotection and remyelination to modify the ALS microenvironment.

Peripheral hyperstimulation alters site of disease onset and course in SOD1 rats

In amyotrophic lateral sclerosis (ALS), the exogenous temporal triggers that result in initial motor neuron death are not understood. Overactivation and consequent accelerated loss of vulnerable motor neurons is one theory of disease initiation. The vulnerability of motor neurons in response to chronic peripheral nerve hyperstimulation was tested in the SOD1G93A rat model of ALS. A novel in vivo technique for peripheral phrenic nerve stimulation was developed via intra-diaphragm muscle electrode implantation at the phrenic motor endpoint. Chronic bilateral phrenic nerve hyperstimulation in SOD1G93A rats accelerated disease progression, including shortened lifespan, hastened motor neuron loss and increased denervation at diaphragm neuromuscular junctions. Hyperstimulation also resulted in focal decline in adjacent forelimb function. These results show that peripheral phrenic nerve hyperstimulation accelerates cell death of vulnerable spinal motor neurons, modifies both temporal and anatomical onset of disease, and leads to involvement of disease in adjacent anatomical regions in this ALS model.

Human neural stem cell grafts in the spinal cord of SOD1 transgenic rats: Differentiation and structural integration into the segmental motor circuitry

Cell replacement strategies for degenerative and traumatic diseases of the nervous system depend on the functional integration of grafted cells into host neural circuitry, a condition necessary for the propagation of physiological signals and, perhaps, targeting of trophic support to injured neurons. We have recently shown that human neural stem cell (NSC) grafts ameliorate motor neuron disease in SOD1 transgenic rodents. Here we study structural aspects of integration of neuronally differentiated human NSCs in the motor circuitry of SOD1 G93A rats. Human NSCs were grafted into the lumbar protuberance of 8-week-old SOD1 G93A rats; the results were compared to those on control Sprague-Dawley rats. Using pre-embedding immuno-electron microscopy, we found human synaptophysin (+) terminals contacting the perikarya and proximal dendrites of host alpha motor neurons. Synaptophysin (+) terminals had well-formed synaptic vesicles and were associated with membrane specializations primarily in the form of symmetrical synapses. To analyze the anatomy of motor circuits engaging differentiated NSCs, we injected the retrograde transneuronal tracer Bartha-pseudorabies virus (PRV) or the retrograde marker cholera toxin B (CTB) into the gastrocnemius muscle/sciatic nerve of SOD1 rats before disease onset and also into control rats. With this tracing, NSC-derived neurons were labeled with PRV but not CTB, a pattern suggesting that PRV entered NSC-derived neurons via transneuronal transfer from host motor neurons but not via direct transport from the host musculature. Our results indicate an advanced degree of structural integration, via functional synapses, of differentiated human NSCs into the segmental motor circuitry of SOD1-G93A rats.

Focal transplantation–based astrocyte replacement is neuroprotective in a model of motor neuron disease

Cellular abnormalities in amyotrophic lateral sclerosis (ALS) are not limited to motor neurons. Astrocyte dysfunction occurs in human ALS and SOD1G93A animal models. Therefore, the value of focal enrichment of normal astrocytes was investigated using transplantation of lineage-restricted astrocyte precursors, Glial-Restricted Precursors (GRPs). GRPs were transplanted around cervical spinal cord respiratory motor neuron pools, the principal cells responsible for death in this neurodegenerative disease. GRPs survived in diseased tissue, differentiated efficiently into astrocytes, and reduced microgliosis in SOD1G93A rat cervical spinal cord. GRPs extended survival and disease duration, attenuated motor neuron loss, and slowed declines in fore-limb motor and respiratory physiological function. Neuroprotection was mediated in part by the primary astrocyte glutamate transporter, GLT1. These findings demonstrate the feasibility and efficacy of transplantation-based astrocyte replacement, and show that targeted multi-segmental cell delivery to cervical spinal cord is a promising therapeutic strategy for slowing focal motor neuron loss associated with ALS.