Gabriela Pintar de Oliveira

Early motor and electrophysiological changes in transgenic mouse model of amyotrophic lateral sclerosis and gender differences on clinical outcome

Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disorder affecting motoneurons and the SOD1(G93A) transgenic mice are widely employed to study disease physiopathology and therapeutic strategies. Despite the cellular and biochemical evidences of an early motor system dysfunction, the conventional behavioral tests do not detect early motor impairments in SOD1 mouse model. We evaluated early changes in motor behavior of ALS mice by doing the analyses of tail elevation, footprint, automatic recording of motor activities by means of an infrared motion sensor activity system and electrophysiological measurements in male and female wild-type (WT) and SOD1(G93A) mice from postnatal day (P) 20 up to endpoint. The classical evaluations of mortality, weight loss, tremor, rotometer, hanging wire and inclined plane were also employed. There was a late onset (after P90) of the impairments of classical parameters and the outcome varied between genders of ALS mice, being tremor, cumulative survival, weight loss and neurological score about 10 days earlier in male than female ALS mice and also about 20 days earlier in ALS males regarding rotarod and hanging wire performances. While diminution of hindpaw base was 10 days earlier in ALS males (P110) compared to females, the steep length decreased 40 days earlier in ALS females (P60) than ALS males. The automatic analysis of motor impairments showed substantial late changes (after P90) of motility and locomotion in the ALS females, but not in the ALS males. It was surprising that the scores of tail elevation were already decreased in ALS males and females by P40, reaching the minimal values at the endpoint. The electrophysiological analyses showed early changes of measures in the ALS mouse sciatic nerve, i.e., decreased values of amplitude (P40) and nerve conduction velocity (P20), and also an increased latency (P20) reaching maximal level of impairments at the late disease phase. The early changes were not accompanied by reductions of neuronal protein markers of neurofilament 200 and ChAT in the ventral part of the lumbar spinal cord of P20 and P60 ALS mice by means of Western blot technique, despite remarkable decreases of those protein levels in P120 ALS mice. In conclusion, early changes of motor behavior and electrophysiological parameters in ALS mouse model must be taken into attention in the analyses of disease mechanisms and therapeutic effects.

Early gene expression changes in spinal cord from SOD1G93A Amyotrophic Lateral Sclerosis animal model

Amyotrophic Lateral Sclerosis (ALS) is an adult-onset and fast progression neurodegenerative disease that leads to the loss of motor neurons. Mechanisms of selective motor neuron loss in ALS are unknown. The early events occurring in the spinal cord that may contribute to motor neuron death are not described, neither astrocytes participation in the pre-symptomatic phases of the disease. In order to identify ALS early events, we performed a microarray analysis employing a whole mouse genome platform to evaluate the gene expression pattern of lumbar spinal cords of transgenic SOD1G93A mice and their littermate controls at pre-symptomatic ages of 40 and 80 days. Differentially expressed genes were identified by means of the Bioconductor packages Agi4×44Preprocess and limma. FunNet web based tool was used for analysis of over-represented pathways. Furthermore, immunolabeled astrocytes from 40 and 80 days old mice were submitted to laser microdissection and RNA was extracted for evaluation of a selected gene by qPCR. Statistical analysis has pointed to 492 differentially expressed genes (155 up and 337 down regulated) in 40 days and 1105 (433 up and 672 down) in 80 days old ALS mice. KEGG analysis demonstrated the over-represented pathways tight junction, antigen processing and presentation, oxidative phosphorylation, endocytosis, chemokine signaling pathway, ubiquitin mediated proteolysis and glutamatergic synapse at both pre-symptomatic ages. Ube2i gene expression was evaluated in astrocytes from both transgenic ages, being up regulated in 40 and 80 days astrocytes enriched samples. Our data points to important early molecular events occurring in pre-symptomatic phases of ALS in mouse model. Early SUMOylation process linked to astrocytes might account to non-autonomous cell toxicity in ALS. Further studies on the signaling pathways presented here may provide new insights to better understand the events triggering motor neuron death in this devastating disorder.

Deregulated expression of cytoskeleton related genes in the spinal cord and sciatic nerve of presymptomatic SOD1G93A Amyotrophic Lateral Sclerosis mouse model

Early molecular events related to cytoskeleton are poorly described in Amyotrophic Lateral Sclerosis (ALS), especially in the Schwann cell (SC), which offers strong trophic support to motor neurons. Database for Annotation, Visualization and Integrated Discovery (DAVID) tool identified cytoskeleton-related genes by employing the Cellular Component Ontology (CCO) in a large gene profiling of lumbar spinal cord and sciatic nerve of presymptomatic SOD1G93A mice. One and five CCO terms related to cytoskeleton were described from the spinal cord deregulated genes of 40 days (actin cytoskeleton) and 80 days (microtubule cytoskeleton, cytoskeleton part, actin cytoskeleton, neurofilament cytoskeleton, and cytoskeleton) old transgene mice, respectively. Also, four terms were depicted from the deregulated genes of sciatic nerve of 60 days old transgenes (actin cytoskeleton, cytoskeleton part, microtubule cytoskeleton and cytoskeleton). Kif1b was the unique deregulated gene in more than one studied region or presymptomatic age. The expression of Kif1b [quantitative polymerase chain reaction (qPCR)] elevated in the lumbar spinal cord (40 days old) and decreased in the sciatic nerve (60 days old) of presymptomatic ALS mice, results that were in line to microarray findings. Upregulation (24.8 fold) of Kif1b was seen in laser microdissected enriched immunolabeled motor neurons from the spinal cord of 40 days old presymptomatic SOD1G93A mice. Furthermore, Kif1b was dowregulated in the sciatic nerve Schwann cells of presymptomatic ALS mice (60 days old) that were enriched by means of cell microdissection (6.35 fold), cell sorting (3.53 fold), and primary culture (2.70 fold) technologies. The gene regulation of cytoskeleton molecules is an important occurrence in motor neurons and Schwann cells in presymptomatic stages of ALS and may be relevant in the dying back mechanisms of neuronal death. Furthermore, a differential regulation of Kif1b in the spinal cord and sciatic nerve cells emerged as key event in ALS.

The importance of molecular histology to study glial influence on neurodegenerative disorders. Focus on recent developed single cell laser microdissection

Neuron-glia interaction is involved in physiological function of neurons, however recent evidences have suggested glial cells as participants in neurotoxic and neurotrophic mechanisms of neurodegenerative/neuroregenerative processes. Histological techniques employing immunolabeling, historadiography and in situ hybridization have been useful to localize at cell levels molecules in normal and pathological situations. The intercellular accomplishment leading to neuronal injury in central nervous system disorders implies the performance of quantitative assays to better interpret the role of related molecules or signal pathways, however one limitation employing the whole tissue is the loss of cellular resolution. The laser capture microdissection was developed recently and allows the selection of specific cell types from their original environment after freezing and sectioning the tissue sampling, leading to the quantification of gene expression in individual cells, thus providing a unique opportunity to get new informations on cell signaling related to neurodegeneration. Here we reviewed the role of glial cell signaling on neurodegenerative disorders like ischemia, Parkinson and Alzheimer diseases, and also amyotrophic lateral sclerosis and what has been published with regards to single cell laser capture microdissection technique in the molecular biology investigation on these issues.

The review of the methods to obtain non-neuronal cells to study glial influence on Amyotrophic Lateral Sclerosis pathophysiology at molecular level in vitro

PURPOSE Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that displays a rapid evolution. Current treatments have failed to revert clinical symptoms because the mechanisms involved in the death of motoneuron are still unknown. Recent publications have put non-neuronal cells, particularly, astrocyte and microglia, in the scenario of pathophisiology of the disease. Animal models for ALS, particularly transgenic mice expressing the human SOD1 gene with a G93A mutation (hSOD1), are available and display the phenotype of the disease at cellular and clinical levels. However, it is a lack of detailed information regarding the methods to study the disease in vitro to better understand the contribution of non-neuronal cells in the onset and progression of the pathology. METHODS Colonies of Swiss mice and transgenic mice expressing hSOD1 mutation as well as non-transgenic controls (wild-type) were amplified after a genotyping evaluation. Disease progression was followed behaviorally and mortality was registered. Highly purified primary cultures of astrocytes and microglia from mouse spinal cord were obtained. Cells were identified by means of GFAP and CD11B immunocytochemistry. The purity of astroglial and microglial cell cultures was also accompanied by means of Western blot and RT-PCR analyses employing a number of markers. RESULTS The disease onset was about 105 days and the majority of transgenic mice displayed the disease symptoms by 125 days of age and reached the endpoint 20 days later. A substantial motor weakens was registered in the transgenic mice compared to wild-type at the end point. Immunocytochemical, biochemical and RT-PCR analyses demonstrated a highly purified primary cultures of spinal cord astrocytes and microglia. CONCLUSION It is possible to achieve highly purified primary cultures of spinal cord astrocytes and microglia to be employed in cellular and molecular analyses of the influence of such non-neuronal cells in the pathophysiology of ALS.

Differential regulation of FGF-2 in neurons and reactive astrocytes of axotomized rat hypoglossal nucleus. A possible therapeutic target for neuroprotection in peripheral nerve pathology.

Despite the favorable treatment of cranial nerve neuropathology in adulthood, some cases are resistant to therapy leading to permanent functional impairments. In many cases, suitable treatment is problematic as the therapeutic target remains unknown. Basic fibroblast growth factor (bFGF, FGF-2) is involved in neuronal maintenance and wound repair following nervous system lesions. It is one of few neurotrophic molecules acting in autocrine, paracrine and intracrine fashions depending upon specific circumstances. Peripheral cranial somatic motor neurons, i.e. hypoglossal (XII) neurons, may offer a unique opportunity to study cellular FGF-2 mechanisms as the molecule is present in the cytoplasm of neurons and in the nuclei of astrocytes of the central nervous system. FGF-2 may trigger differential actions during development, maintenance and lesion of XII neurons because axotomy of those cells leads to cell death during neonatal ages, but not in adult life. Moreover, the modulatory effects of astroglial FGF-2 and the Ca+2-binding protein S100β have been postulated in paracrine mechanisms after neuronal lesions. In our study, adult Wistar rats received a unilateral crush or transection (with amputation of stumps) of XII nerve, and were sacrificed after 72h or 11 days. Brains were processed for immunohistochemical localization of neurofilaments (NF), with or without counterstaining for Nissl substance, glial fibrillary acidic protein (GFAP, as a marker of astrocytes), S100β and FGF-2. The number of Nissl-positive neurons of axotomized XII nucleus did not differ from controls. The NF immunoreactivity increased in the perikarya and decreased in the neuropil of axotomized XII neurons 11 days after nerve crush or transection. An astrocytic reaction was seen in the ipsilateral XII nucleus of the crushed or transected animals 72h and 11 days after the surgery. The nerve lesions did not change the number of FGF-2 neurons in the ipsilateral XII nucleus; however, the nerve transection increased the number of FGF-2 glial profiles by 72h and 11 days. Microdensitometric image analysis revealed a short lasting decrease in the intensity of FGF-2 immunoreactivity in axotomized XII neurons by 72h after nerve crush or transection and also an elevation of FGF-2 in the ipsilateral of glial nuclei by 72h and 11 days after the two lesions. S100β decreased in astrocytes of 11-day-transected XII nucleus. The two-color immunoperoxidase for the simultaneous detection of the GFAP/FGF-2 indicated FGF-2 upregulation in the nuclei of reactive astrocytes of the lesioned XII nucleus. Astroglial FGF-2 may exert paracrine trophic actions in mature axotomized XII neurons and might represent a therapeutic target for neuroprotection in peripheral nerve pathology.

A method to immunolabel rodent spinal cord neurons and glia for molecular study in specific laser microdissected cells involved in neurodegenerative disorders

Neuron-glia interaction is involved in physiological function of neurons, however, recent evidences have suggested glial cells as participants in neurotoxic and neurotrophic mechanisms of neurodegenerative/neuroregenerative processes. Laser microdissection offers a unique opportunity to study molecular regulation in specific immunolabeled cell types. However, an adequate protocol to allow morphological and molecular analysis of rodent spinal cord astrocyte, microglia and motoneurons remains a big challenge. In this paper we present a quick method to immunolabel those cells in flash frozen sections to be used in molecular biology analyses after laser microdissection and pressure catapulting.

Behavioral Improvement and Regulation of Molecules Related to Neuroplasticity in Ischemic Rat Spinal Cord Treated with PEDF

Pigment epithelium derived factor (PEDF) exerts trophic actions to motoneurons and modulates nonneuronal restorative events, but its effects on neuroplasticity responses after spinal cord (SC) injury are unknown. Rats received a low thoracic SC photothrombotic ischemia and local injection of PEDF and were evaluated behaviorally six weeks later. PEDF actions were detailed in SC ventral horn (motor) in the levels of the lumbar central pattern generator (CPG), far from the injury site. Molecules related to neuroplasticity (MAP-2), those that are able to modulate such event, for instance, neurotrophic factors (NT-3, GDNF, BDNF, and FGF-2), chondroitin sulfate proteoglycans (CSPG), and those associated with angiogenesis and antiapoptosis (laminin and Bcl-2) and Eph (receptor)/ephrin system were evaluated at cellular or molecular levels. PEDF injection improved motor behavioral performance and increased MAP-2 levels and dendritic processes in the region of lumbar CPG. Treatment also elevated GDNF and decreased NT-3, laminin, and CSPG. Injury elevated EphA4 and ephrin-B1 levels, and PEDF treatment increased ephrin A2 and ephrins B1, B2, and B3. Eph receptors and ephrins were found in specific populations of neurons and astrocytes. PEDF treatment to SC injury triggered neuroplasticity in lumbar CPG and regulation of neurotrophic factors, extracellular matrix molecules, and ephrins.

Evaluation of Neuronal Apoptosis Precursors in an Experimental Model of Acute Normovolemic Hemodilution

Background The effects of acute anemia on neuronal cells and the safe limits of hematocrit are not well established. The objective of this study was to evaluate neuronal pro- and anti-apoptotic Bax and Bcl-x proteins, caspase-3 and -9 activity, and DNA fragmentation after acute normovolemic hemodilution (ANH). Methods Twenty-four pigs were anesthetized and randomized into 4 groups: Sham, ANH to 15% hematocrit (ANH15%), ANH to 10% hematocrit (ANH10%) and hypoxia (Hx). ANH was achieved by simultaneous blood withdrawal and hydroxyethyl starch infusion. Hx consisted of ventilation with a 6% inspired oxygen fraction for 60 minutes. Bax and Bcl-x proteins as well as DNA fragmentation were evaluated in cortical nuclear and mitochondrial fractions. Caspase-3 and -9 activity was evaluated in the cortical mitochondrial and hippocampal cytosolic fractions. The data were compared using analysis of variance followed by Tukey’s test (P<0.05). Results No changes were observed in Bax protein expression after hemodilution in the ANH15% and ANH10% groups compared to the Sham group. Bax expression in the Hx group was increased in the nuclear and mitochondrial fractions compared to all other groups. No significant difference was observed in Bcl-x expression. Caspase-3 and -9 activity in the cytosolic and mitochondrial fractions was different in the Hx group compared to all other groups. No statistical significance in DNA fragmentation was found among the Sham, ANH15% or ANH10% groups. Conclusion ANH to 10 and 15% hematocrit did not induce alterations in apoptosis precursors, suggesting that cerebral oxygenation was preserved during these anemic states.

Differential cellular FGF-2 upregulation in the rat facial nucleus following axotomy, functional electrical stimulation and corticosterone: a possible therapeutic target to Bell's palsy

Background The etiology of Bells palsy can vary but anterograde axonal degeneration may delay spontaneous functional recovery leading the necessity of therapeutic interventions. Corticotherapy and/or complementary rehabilitation interventions have been employed. Thus the natural history of the disease reports to a neurotrophic resistance of adult facial motoneurons leading a favorable evolution however the related molecular mechanisms that might be therapeutically addressed in the resistant cases are not known. Fibroblast growth factor-2 (FGF-2) pathway signaling is a potential candidate for therapeutic development because its role on wound repair and autocrine/paracrine trophic mechanisms in the lesioned nervous system. Methods Adult rats received unilateral facial nerve crush, transection with amputation of nerve branches, or sham operation. Other group of unlesioned rats received a daily functional electrical stimulation in the levator labii superioris muscle (1 mA, 30 Hz, square wave) or systemic corticosterone (10 mgkg-1). Animals were sacrificed seven days later. Results Crush and transection lesions promoted no changes in the number of neurons but increased the neurofilament in the neuronal neuropil of axotomized facial nuclei. Axotomy also elevated the number of GFAP astrocytes (143% after crush; 277% after transection) and nuclear FGF-2 (57% after transection) in astrocytes (confirmed by two-color immunoperoxidase) in the ipsilateral facial nucleus. Image analysis reveled that a seven days functional electrical stimulation or corticosterone led to elevations of FGF-2 in the cytoplasm of neurons and in the nucleus of reactive astrocytes, respectively, without astrocytic reaction. Conclusion FGF-2 may exert paracrine/autocrine trophic actions in the facial nucleus and may be relevant as a therapeutic target to Bells palsy.