Animal Models Of Neurological Disorders Research Articles

Glial cell line-derived neurotrophic factor-secreting human neural progenitors show long-term survival, maturation into astrocytes, and no tumor formation following transplantation into the spinal cord of immunocompromised rats

Human neural progenitor cells (hNPCs) derived from the fetal cortex can be expanded in vitro and genetically modified through lentiviral transduction to secrete growth factors shown to have a neurotrophic effect in animal models of neurological disease. hNPCs survive and mature following transplantation into the central nervous system of large and small animals including the rat model of amyotrophic lateral sclerosis. Here we report that hNPCs engineered to express glial cell line-derived neurotrophic factor (GDNF) survive long-term (7.5 months) following transplantation into the spinal cord of athymic nude rats and continue to secrete GDNF. Cell proliferation declined while the number of astrocytes increased, suggesting final maturation of the cells over time in vivo. Together these data show that GDNF-producing hNPCs may be useful as a source of cells for long-term delivery of both astrocytes and GDNF to the damaged central nervous system.

Metformin as a key to alternative activation of microglia?

The results of recent studies suggest that metformin, in addition to its antihyperglycemic efficacy, may also attenuate neuroinflammation and directly act on the central nervous system. However, the molecular mechanisms by which metformin exerts its anti-inflammatory effects in the brain remain largely unknown. Adenosine-monophosphate-activated protein kinase (AMPK) activation is the most well-known mechanism of metformin action. However, some of the biological responses to metformin (e.g. the release of cytokines and the expression of arginase I or PGC-1α) are not limited to AMPK activation but also are mediated by AMPK-independent mechanisms. This article reviews current evidence supporting the hypothesis that the shift of microglia toward alternative activation may underlie the beneficial effects of metformin observed in animal models of neurological disorders.

Probenecid: An Emerging Tool for Neuroprotection

Probenecid (PROB) has been widely used for long time for different clinical purposes, from gout treatment to designs as a coadjutant for antibiotic agents. Among its many properties, the ability of PROB to preserve high concentrations of several metabolites and other agents in the CNS, together with its relative lack of side-effects, have made this drug a valuable pharmacological tool for clinical and basic research. Nowadays, biomedical research offers evidence about new targets for PROB that may help to explain its many beneficial actions. In this regard, despite most of its protective actions in the brain have been largely related to its capacity to accumulate the inhibitory metabolite kynurenic acid to further inhibit the glutamate-related excitotoxicity in different animal models of neurological disorders, in this review we describe the basic aspects of PROB's pharmacokinetics and mechanisms of action and discuss other alternative targets recently described for this drug that may complement its pattern of activity in the CNS, including its role as anti-inflammatory and anti-nociceptive agent when targeting different key proteins.

Modulation of nitrergic signalling pathway by American ginseng attenuates chronic unpredictable stress-induced cognitive impairment, neuroinflammation, and biochemical alterations

Prolonged stress causes extensive loss of neurons leading to deficits in cognitive performance. Increasing evidence indicates that accumulation of intercellular messenger, nitric oxide (NO), plays a crucial role in the pathogenesis of memory disorders. American ginseng (AG) is known to show protection in different animal models of neurological diseases; however, its exact mechanism of action is not clearly understood. Therefore, the current study was designed to investigate the interaction of AG against chronic unpredictable stress (CUS)-associated behavioral and biochemical alterations and the probable role of nitrergic pathway in this effect. Male Laca mice were exposed to a series of stressors along with drug/vehicle treatment daily for 28days. CUS paradigm caused significant impairment in both acquisition and retention memory as measured in Morris water maze and elevated plus maze task. This was coupled with alterations in oxidative stress markers, mitochondrial enzyme complex activities, pro-inflammatory cytokine (TNF-α), and acetylcholinesterase levels in the hippocampus as compared with naïve group. Besides, there was a marked increase in serum corticosterone levels. AG (100, 200mg/kg; p.o.) treatment significantly improved cognitive impairment; reduced TNF-α, acetylcholinesterase, and corticosterone levels; and attenuated oxidative-nitrergic stress. Furthermore, pre-treatment of L-arginine (100mg/kg; i.p.), a nitric oxide donor, with subeffective dose of AG (100mg/kg; p.o.) reversed its protective effects. However, L-NAME (10mg/kg, i.p.), a non-specific NO synthase inhibitor, potentiated the effects of AG. Our findings suggest that modulation of nitrergic signalling cascade is involved in the protective effects of AG against CUS-induced cognitive dysfunction, oxidative stress, and neuroinflammation.

Improved Method of Producing Human Neural Progenitor Cells of High Purity and in Large Quantities from Pluripotent Stem Cells for Transplantation Studies

Transplantation of human neural progenitor cells (hNPCs) is a promising therapeutic approach for various diseases of the central nervous system (CNS). Reliable testing of hNPC transplantation in animal models of neurological diseases requires that these cells can be produced in sufficient amounts, show consistent homogeneity as a neural cell population, and be reliably labeled for in vivo tracking. In addition, the cells should be characterized as being at the optimal state of differentiation favoring successful engraftment. Here, we show that high numbers of purified hNPCs can be produced from human embryonic stem cells (hESCs) by manually selecting specifically sized and shaped spheres followed by fluorescence-activated cell sorting based on the relative cell size. In addition, we report that labeling of hNPCs with ultra-small superparamagnetic iron oxide (USPIO) particles does not affect the cellular morphology or growth. More importantly, we show that the transduction with lentiviral vector encoding green fluorescent protein (GFP) decreases the neurality of the cell population. We conclude that our cost-effective protocol of generating hNPCs is widely applicable for preclinical studies on CNS disorders. This improved method of producing large quantities of high-purity hNPCs maybe useful also when generating hNPCs from human induced pluripotent stem (hiPS) cell lines. However, caution should be used when lenti-GFP transduction is applied for hNPC labeling.

Class I Histone Deacetylase Inhibitor Valproic Acid Reverses Cognitive Deficits in a Mouse Model of Septic Encephalopathy

Accumulating evidence suggests that histone deacetylase inhibitor exert neuroprotective effects in animal models of neurological diseases. We investigated for the first time whether class I histone deacetylase inhibitor valproic acid (VPA) can reverse cognitive deficits in a mouse model of sepsis-associated encephalopathy (SAE). Moreover, the possible mechanisms of protection were also explored. A mouse model of SAE was induced in adult male mice by cecal ligation and puncture (CLP). Mice received an administration of saline or VPA (100 mg/kg) once daily for 14 consecutive days starting either immediately or 2 weeks after operation. Furthermore, the TrkB antagonist K252a was used in another group of experiment to investigate whether brain-derived neurotrophic factor (BDNF)-TrkB signaling pathway is involved in the protection of VPA. Our data suggested that CLP resulted in significant cognitive impairments accompanied by increased expressions in interleukin-1β and caspase-3, and decreased expressions in BDNF, phospho-TrkB (pTrkB), postsynaptic density 95, and synapses, which were reversed by VPA. However, TrkB antagonist K252a abolished the beneficial effects of VPA with regard to cognition and decreased pTrkB expression and synapses in the hippocampus. Taken together, the findings of the present study suggested chronic treatment with VPA reverses cognitive deficits through mechanisms probably via a reduction in inflammation and apoptosis in the brain, as well as the activation of the BDNF-TrkB signaling pathway in a mouse model of SAE.

Acetyltransferases (HATs) as targets for neurological therapeutics.

The acetylation of histone and non-histone proteins controls a great deal of cellular functions, thereby affecting the entire organism, including the brain. Acetylation modifications are mediated through histone acetyltransferases (HAT) and deacetylases (HDAC), and the balance of these enzymes regulates neuronal homeostasis, maintaining the pre-existing acetyl marks responsible for the global chromatin structure, as well as regulating specific dynamic acetyl marks that respond to changes and facilitate neurons to encode and strengthen long-term events in the brain circuitry (e.g., memory formation). Unfortunately, the dysfunction of these finely-tuned regulations might lead to pathological conditions, and the deregulation of the HAT/HDAC balance has been implicated in neurological disorders. During the last decade, research has focused on HDAC inhibitors that induce a histone hyperacetylated state to compensate acetylation deficits. The use of these inhibitors as a therapeutic option was efficient in several animal models of neurological disorders. The elaboration of new cell-permeant HAT activators opens a new era of research on acetylation regulation. Although pathological animal models have not been tested yet, HAT activator molecules have already proven to be beneficial in ameliorating brain functions associated with learning and memory, and adult neurogenesis in wild-type animals. Thus, HAT activator molecules contribute to an exciting area of research.

Therapeutic Effects of Progesterone in Animal Models of Neurological Disorders

Substantial evidence supports that progesterone exerts many functions in the central and peripheral nervous system unrelated to its classical role in reproduction. In this review we first discussed progesterone effects following binding to the classical intracellular progesterone receptors A and B and several forms of membrane progesterone receptors, the modulation of intracellular signalling cascades and the interaction of progesterone reduced metabolites with neurotransmitter receptors. We next described our results involving animal models of human neuropathologies to elucidate the protective roles of progesterone. We described: (a) the protective and promyelinating effects of progesterone in experimental spinal cord injury; (b) the progesterone protective effects exerted upon motoneurons in the degenerating spinal cord of Wobbler mouse model of amyotropic lateral sclerosis; (c) the protective and anti-inflammatory effects of progesterone in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis and after lysolecithin demyelination; (d) the progesterone prevention of nociception and neuropathic pain which follow spinal cord injury; and (e) the protective effect of progesterone in experimental ischemic stroke. Whenever available, the molecular mechanisms involved in these progesterone effects were examined. The multiplicity of progesterone beneficial effects has opened new venues of research for neurological disorders. In this way, results obtained in animal models could provide the basis for novel therapeutic strategies and pre-clinical studies.

Bioanalytical method for evaluating the pharmacokinetics of the GCP-II inhibitor 2-phosphonomethyl pentanedioic acid (2-PMPA)

2-Phosphonomethyl pentanedioic acid (2-PMPA) is a potent and selective inhibitor of glutamate carboxypeptidase-II, an enzyme which catabolizes the abundant neuropeptide N-acetyl-aspartyl-glutamate (NAAG) to N-acetylaspartate (NAA) and glutamate. 2-PMPA demonstrates robust efficacy in numerous animal models of neurological disease, however its pharmacokinetics has not yet been fully described. 2-PMPA is a highly polar compound with multiple negative charges causing significant challenges for analysis in biological matrices. Here we report a derivatization method for the acidic groups that involved protein precipitation with acetonitrile followed by reaction with N-tert-butyldimethysilyl-N-methyltrifluoroacetamide (MTBSTFA). The silylated analyte with transitions (683→551.4) and the internal standard (669→537.2) were monitored by tandem mass spectrometry with electrospray positive ionization mode. The method was subsequently used to evaluate 2-PMPA pharmacokinetics in rats. Intraperitoneal administration of 100mg/kg 2-PMPA resulted in maximum concentration in plasma of 275μg/mL at 0.25h. The half-life, area under the curve, apparent clearance, and volume of distribution were 0.64h, 210μg×h/mL, 7.93mL/min/kg, and 0.44L/kg, respectively. The tissue/plasma ratios in brain, sciatic nerve and dorsal root ganglion were 0.018, 0.120 and 0.142, respectively. In summary, a sensitive analytical method for 2-PMPA is reported that can be employed for similarly charged molecules.

Progress in Stem Cell Therapy for Major Human Neurological Disorders

Human neurological disorders such as Alzheimer's disease (AD), Parkinson's disease, stroke or spinal cord injury are caused by the loss of neurons and glial cells in the brain or spinal cord in the Central Nervous System (CNS). Stem cell technology has become an attractive option to investigate and treat these diseases. Several types of neurons and glial cells have successfully been generated from stem cells, which in some cases, have ameliorated some dysfunctions both in animal models of neurological disorders and in patients at clinical level. Stem cell-based therapies can be beneficial by acting through several mechanisms such as cell replacement, modulation of inflammation and trophic actions. Here we review recent and current remarkable clinical studies involving stem cell-based therapy for AD and stroke and provide an overview of the different types of stem cells available nowadays, their main properties and how they are developing as a possible therapy for neurological disorders.

The Battle of the Sexes for Stroke Therapy: Female- Versus Male-Derived Stem Cells

Cell therapy is a major discipline of regenerative medicine that has been continually growing over the last two decades. The aging of the population necessitates discovery of therapeutic innovations to combat debilitating disorders, such as stroke. Menstrual blood and Sertoli cells are two gender-specific sources of viable transplantable cells for stroke therapy. The use of autologous cells for the subacute phase of stroke offers practical clinical application. Menstrual blood cells are readily available, display proliferative capacity, pluripotency and angiogenic features, and, following transplantation in stroke models, have the ability to migrate to the infarct site, regulate the inflammatory response, secrete neurotrophic factors, and have the possibility to differentiate into neural lineage. Similarly, the testis-derived Sertoli cells secrete many growth and trophic factors, are highly immunosuppressive, and exert neuroprotective effects in animal models of neurological disorders. We highlight the practicality of experimental and clinical application of menstrual blood cells and Sertoli cells to treat stroke, from cell isolation and cryopreservation to administration.

A Micro-Silicon Chip for in Vivo Cerebral Imprint in Monkey

Access to cerebral tissue is essential to better understand the molecular mechanisms associated with neurodegenerative diseases. In this study, we present, for the first time, a new tool designed to obtain molecular and cellular cerebral imprints in the striatum of anesthetized monkeys. The imprint is obtained during a spatially controlled interaction of a chemically modified micro-silicon chip with the brain tissue. Scanning electron and immunofluorescence microscopies showed homogeneous capture of cerebral tissue. Nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) analysis of proteins harvested on the chip allowed the identification of 1158 different species of proteins. The gene expression profiles of mRNA extracted from the imprint tool showed great similarity to those obtained via the gold standard approach, which is based on post-mortem sections of the same nucleus. Functional analysis of the harvested molecules confirmed the spatially controlled capture of striatal proteins implicated in dopaminergic regulation. Finally, the behavioral monitoring and histological results establish the safety of obtaining repeated cerebral imprints in striatal regions. These results demonstrate the ability of our imprint tool to explore the molecular content of deep brain regions in vivo. They open the way to the molecular exploration of brain in animal models of neurological diseases and will provide complementary information to current data mainly restricted to post-mortem samples.

Modulation of adult-born neurons in the inflamed hippocampus

Throughout life new neurons are continuously added to the hippocampal circuitry involved with spatial learning and memory. These new cells originate from neural precursors in the subgranular zone of the dentate gyrus, migrate into the granule cell layer, and integrate into neural networks encoding spatial and contextual information. This process can be influenced by several environmental and endogenous factors and is modified in different animal models of neurological disorders. Neuroinflammation, as defined by the presence of activated microglia, is a common key factor to the progression of neurological disorders. Analysis of the literature shows that microglial activation impacts not only the production, but also the migration and the recruitment of new neurons. The impact of microglia on adult-born neurons appears much more multifaceted than ever envisioned before, combining both supportive and detrimental effects that are dependent upon the activation phenotype and the factors being released. The development of strategies aimed to change microglia toward states that promote functional neurogenesis could therefore offer novel therapeutic opportunities against neurological disorders associated with cognitive deficits and neuroinflammation. The present review summarizes the current knowledge on how production, distribution, and recruitment of new neurons into behaviorally relevant neural networks are modified in the inflamed hippocampus.

Selective Gelatinase Inhibitor Neuroprotective Agents Cross the Blood-Brain Barrier

SB-3CT, a potent and selective inhibitor of matrix metalloproteinase-2 and -9, has shown efficacy in several animal models of neurological diseases. One of the greatest challenges in the development of therapeutics for neurological diseases is the inability of drugs to cross the blood-brain barrier. A sensitive bioanalytical method based on ultraperformance liquid chromatography with multiple-reaction monitoring detection was developed to measure levels of SB-3CT, its active metabolite, the α-methyl analogue, and its p-hydroxy metabolite in plasma and brain. The compounds are rapidly absorbed and are readily distributed to the brain. The pharmacokinetic properties of these gelatinase inhibitors and the efficacy shown by SB-3CT in animal models of stroke, subarachnoid hemorrhage, and spinal cord injury indicate that this class of compounds holds considerable promise in the treatment of diseases of the central nervous system.

Cell Therapy Modulates Expression of Tax1-Binding Protein 1 and Synaptotagmin IV in a Model of Optic Nerve Lesion

Bone marrow mononuclear cells (BMMCs) have been used with considerable success to improve regeneration and/or functional recovery in animal models of neurologic diseases. Injected into the host, they migrate to the damaged areas and release cytokines and/or trophic factors, which are capable of altering the genetic program of the injured tissue cells. In this study, there was a search for genes with altered expression in a model of optic nerve crush and cell therapy. Optic nerve crush was followed by an intravitreous injection of BMMCs or vehicle in adult rats. After 14 days, we obtained a transcriptome screening of the retinas using differential display and automatic sequencing, followed by q-PCR, Western blot, and immunohistochemistry of selected genes and proteins. Among the differentially displayed genes, transcription of the antiapoptotic Tax1-binding protein 1 (Tax1BP1) and Synaptotagmin IV (Syt IV), an immediate early gene, is increased in the treated group. Tax1BP1 expression is robust in the ganglion cell layer and is significantly increased by cell therapy. Syt IV is expressed by activated Müller cells and astrocytes in the retina and optic nerve, without changes in protein levels among the groups. Tax1BP1 and Syt IV transcription and/or expression are differently modulated by optic nerve crush and BMMC treatment, and might be related to neuronal damage and cell-therapy effects in the retina. The increased expression of Tax1BP1 in the treated eyes could be involved in the neuroprotective effects of BMMCs that were described previously by our group.

Effects of aging on behavioral assessment performance: implications for clinically relevant models of neurological disease

Despite the role of aging in development of neurological and neurodegenerative diseases, the effects of age are often disregarded in experimental design of preclinical studies. Functional assessment increases the clinical relevance of animal models of neurological disease and adds value beyond traditional histological measures. However, the relationship between age and functional impairment has not been systematically assessed through a battery of functional tests. In this study, various sensorimotor and behavioral tests were used to evaluate effects of aging on functional performance in naive animals. Sensorimotor measures included locomotor activity; Rotarod, inclined plane, and grip-strength testing; and modified Neurological Severity Score. The Morris water maze was used to examine differences in learning and memory, and the elevated plus maze and forced swim test were used to assess anxiety-like and depressive-like behaviors, respectively. Older Sprague-Dawley rats (18-20 months) were found to perform significantly worse on the inclined plane tests, and they exhibited alterations in elevated-plus maze and forced swim test compared with young adult rats (3-4 months). Specifically, older rats exhibited reduced exploration of open arms in elevated plus maze and higher immobility time in forced swim test. Spatial acquisition and reference memory were diminished in older rats compared with those in young adult rats. This study demonstrates clear differences between naive young adult and older animals, which may have implications in functional assessment for preclinical models of neurological disease.

Identification of the Rostral Migratory Stream in the Canine and Feline Brain

In the adult rodent brain, neural progenitor cells migrate from the subventricular zone of the lateral ventricle towards the olfactory bulb in a track known as the rostral migratory stream (RMS). To facilitate the study of neural progenitor cells and stem cell therapy in large animal models of CNS disease, we now report the location and characteristics of the normal canine and feline RMS. The RMS was found in Nissl-stained sagittal sections of adult canine and feline brains as a prominent, dense, continuous cellular track beginning at the base of the anterior horn of the lateral ventricle, curving around the head of the caudate nucleus and continuing laterally and ventrally to the olfactory peduncle before entering the olfactory tract and bulb. To determine if cells in the RMS were proliferating, the thymidine analog 5-bromo-2-deoxyuridine (BrdU) was administered and detected by immunostaining. BrdU-immunoreactive cells were present throughout this track. The RMS was also immunoreactive for markers of proliferating cells, progenitor cells and immature neurons (Ki-67 and doublecortin), but not for NeuN, a marker of mature neurons. Luxol fast blue and CNPase staining indicated that myelin is closely apposed to the RMS along much of its length and may provide guidance cues for the migrating cells. Identification and characterization of the RMS in canine and feline brain will facilitate studies of neural progenitor cell biology and migration in large animal models of neurologic disease.

In Vitro Generation of Three‐Dimensional Substrate‐Adherent Embryonic Stem Cell–Derived Neural Aggregates for Application in Animal Models of Neurological Disorders

In vitro-differentiated embryonic stem (ES) cells comprise a useful source for cell replacement therapy, but the efficiency and safety of a translational approach are highly dependent on optimized protocols for directed differentiation of ES cells into the desired cell types in vitro. Furthermore, the transplantation of three-dimensional ES cell-derived structures instead of a single-cell suspension may improve graft survival and function by providing a beneficial microenvironment for implanted cells. To this end, we have developed a new method to efficiently differentiate mouse ES cells into neural aggregates that consist predominantly (>90%) of postmitotic neurons, neural progenitor cells, and radial glia-like cells. When transplanted into the excitotoxically lesioned striatum of adult mice, these substrate-adherent embryonic stem cell-derived neural aggregates (SENAs) showed significant advantages over transplanted single-cell suspensions of ES cell-derived neural cells, including improved survival of GABAergic neurons, increased cell migration, and significantly decreased risk of teratoma formation. Furthermore, SENAs mediated functional improvement after transplantation into animal models of Parkinson's disease and spinal cord injury. This unit describes in detail how SENAs are efficiently derived from mouse ES cells in vitro and how SENAs are isolated for transplantation. Furthermore, methods are presented for successful implantation of SENAs into animal models of Huntington's disease, Parkinson's disease, and spinal cord injury to study the effects of stem cell-derived neural aggregates in a disease context in vivo.

Specific In Vivo Staining of Astrocytes in the Whole Brain after Intravenous Injection of Sulforhodamine Dyes

Fluorescent staining of astrocytes without damaging or interfering with normal brain functions is essential for intravital microscopy studies. Current methods involved either transgenic mice or local intracerebral injection of sulforhodamine 101. Transgenic rat models rarely exist, and in mice, a backcross with GFAP transgenic mice may be difficult. Local injections of fluorescent dyes are invasive. Here, we propose a non-invasive, specific and ubiquitous method to stain astrocytes in vivo. This method is based on iv injection of sulforhodamine dyes and is applicable on rats and mice from postnatal age to adulthood. The astrocytes staining obtained after iv injection was maintained for nearly half a day and showed no adverse reaction on astrocytic calcium signals or electroencephalographic recordings in vivo. The high contrast of the staining facilitates the image processing and allows to quantify 3D morphological parameters of the astrocytes and to characterize their network. Our method may become a reference for in vivo staining of the whole astrocytes population in animal models of neurological disorders.

CHARACTERIZATION OF MOLECULAR CHANGES IN CSF OF PATIENTS WITH RECENT ONSET SCHIZOPHRENIA

2-Phosphonomethyl pentanedioic acid (2-PMPA) is a potent and selective inhibitor of glutamate carboxypeptidase-II, an enzyme which catabolizes the abundant neuropeptide N-acetyl-aspartyl-glutamate (NAAG) to N-acetylaspartate (NAA) and glutamate. 2-PMPA demonstrates robust efficacy in numerous animal models of neurological disease, however its pharmacokinetics has not yet been fully described. 2-PMPA is a highly polar compound with multiple negative charges causing significant challenges for analysis in biological matrices. Here we report a derivatization method for the acidic groups that involved protein precipitation with acetonitrile followed by reaction with N-tert-butyldimethysilyl-N-methyltrifluoroacetamide (MTBSTFA). The silylated analyte with transitions (683→551.4) and the internal standard (669→537.2) were monitored by tandem mass spectrometry with electrospray positive ionization mode. The method was subsequently used to evaluate 2-PMPA pharmacokinetics in rats. Intraperitoneal administration of 100 mg/kg 2-PMPA resulted in maximum concentration in plasma of 275 μg/mL at 0.25 h. The half-life, area under the curve, apparent clearance, and volume of distribution were 0.64 h, 210 μg × h/mL, 7.93 mL/min/kg, and 0.44 L/kg, respectively. The tissue/plasma ratios in brain, sciatic nerve and dorsal root ganglion were 0.018, 0.120 and 0.142, respectively. In summary, a sensitive analytical method for 2-PMPA is reported that can be employed for similarly charged molecules.