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1.
PLoS One ; 10(2): e0116594, 2015.
Article in English | MEDLINE | ID: mdl-25647322

ABSTRACT

Ornithine transcarbamylase deficiency (OTCD, OMIM# 311250) is an inherited X-linked urea cycle disorder that is characterized by hyperammonemia and orotic aciduria. In this report, we describe a new animal model of OTCD caused by a spontaneous mutation in the mouse Otc gene (c.240T>A, p.K80N). This transversion in exon 3 of ornithine transcarbamylase leads to normal levels of mRNA with low levels of mature protein and is homologous to a mutation that has also been described in a single patient affected with late-onset OTCD. With higher residual enzyme activity, spf-J were found to have normal plasma ammonia and orotate. Baseline plasma amino acid profiles were consistent with mild OTCD: elevated glutamine, and lower citrulline and arginine. In contrast to WT, spf-J displayed baseline elevations in cerebral amino acids with depletion following immune challenge with polyinosinic:polycytidylic acid. Our results indicate that the mild spf-J mutation constitutes a new mouse model that is suitable for mechanistic studies of mild OTCD and the exploration of cerebral pathophysiology during acute decompensation that characterizes proximal urea cycle dysfunction in humans.


Subject(s)
Amino Acids/metabolism , Brain/metabolism , Ornithine Carbamoyltransferase Deficiency Disease/immunology , Ornithine Carbamoyltransferase Deficiency Disease/metabolism , Amino Acid Sequence , Animals , Biological Transport , Body Weight , Brain/drug effects , Disease Models, Animal , Humans , Mice , Molecular Sequence Data , Mutation, Missense , Ornithine Carbamoyltransferase/chemistry , Ornithine Carbamoyltransferase/genetics , Ornithine Carbamoyltransferase/metabolism , Ornithine Carbamoyltransferase Deficiency Disease/genetics , Orotic Acid/metabolism , Phenotype , Poly I-C/pharmacology , Protein Structure, Tertiary , Rats , Survival Analysis
2.
Neural Regen Res ; 9(24): 2164-73, 2014 Dec 15.
Article in English | MEDLINE | ID: mdl-25657738

ABSTRACT

The spinal cord has the ability to regenerate but the microenvironment generated after trauma reduces that capacity. An increase in Src family kinase (SFK) activity has been implicated in neuropathological conditions associated with central nervous system trauma. Therefore, we hypothesized that a decrease in SFK activation by a long-term treatment with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine (PP2), a selective SFK inhibitor, after spinal cord contusion with the New York University (NYU) impactor device would generate a permissive environment that improves axonal sprouting and/or behavioral activity. Results demonstrated that long-term blockade of SFK activation with PP2 increases locomotor activity at 7, 14, 21 and 28 days post-injury in the Basso, Beattie, and Bresnahan open field test, round and square beam crossing tests. In addition, an increase in white matter spared tissue and serotonin fiber density was observed in animals treated with PP2. However, blockade of SFK activity did not change the astrocytic response or infiltration of cells from the immune system at 28 days post-injury. Moreover, a reduced SFK activity with PP2 diminished Ephexin (a guanine nucleotide exchange factor) phosphorylation in the acute phase (4 days post-injury) after trauma. Together, these findings suggest a potential role of SFK in the regulation of spared tissue and/or axonal outgrowth that may result in functional locomotor recovery during the pathophysiology generated after spinal cord injury. Our study also points out that ephexin1 phosphorylation (activation) by SFK action may be involved in the repulsive microenvironment generated after spinal cord injury.

3.
J Mol Neurosci ; 49(2): 347-59, 2013 Feb.
Article in English | MEDLINE | ID: mdl-22878913

ABSTRACT

Some receptors that block axonal regeneration or promote cell death after spinal cord injury (SCI) are localized in membrane rafts. Flotillin-2 (Flot-2) is an essential protein associated with the formation of these domains and the clustering of membranal proteins, which may have signaling activities. Our hypothesis is that trauma will change Flot-2 expression and interference of this lipid raft marker will promote functional locomotor recovery after SCI. Analyses were conducted to determine the spatiotemporal profile of Flot-2 expression in adult rats after SCI, using the MASCIS impactor device. Immunoblots showed that SCI produced a significant decrease in the level of Flot-2 at 2 days post-injury (DPI) that increased until 28 DPI. Confocal microscopy revealed Flot-2 expression in neurons, reactive astrocytes and oligodendrocytes specifically associated to myelin structures near or close to the axons of the cord. In the open field test and grid walking assays, to monitor locomotor recovery of injured rats infused intrathecally with Flot-2 antisense oligonucleotides for 28 days showed significant behavioral improvement at 14, 21 and 28 DPI. These findings suggest that Flot-2 has a role in the nonpermissive environment that blocks locomotor recovery after SCI by clustering unfavorable proteins in membrane rafts.


Subject(s)
Membrane Proteins/metabolism , Spinal Cord Injuries/metabolism , Animals , Astrocytes/metabolism , Female , Gene Expression , Membrane Microdomains/metabolism , Membrane Proteins/genetics , Motor Activity , Myelin Sheath/metabolism , Neurons/metabolism , Rats , Rats, Sprague-Dawley , Spinal Cord/cytology , Spinal Cord/metabolism , Spinal Cord Injuries/physiopathology
4.
J Mol Neurosci ; 46(1): 167-76, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21647706

ABSTRACT

Spinal cord injury (SCI) triggers a sequel of events commonly associated with cell death and dysfunction of glias and neurons surrounding the lesion. Although astrogliosis and glial scar formation have been involved in both damage and repair processes after SCI, their role remains controversial. Our goal was to investigate the effects of the P2 receptors antagonists, PPADS and suramin, in the establishment of the reactive gliosis and the formation of the glial scar. Molecular biology, immunohistochemistry, spared tissue, and locomotor behavioral studies were used to evaluate astrogliosis, in adult female Sprague-Dawley rats treated with P2 antagonists after moderate injury with the NYU impactor device. Semi-quantitative RT-PCR confirmed the presence of P2Y(1,) P2Y(2,) P2Y(4,) P2Y(6,) P2Y(12), and P2X(2) receptors in the adult spinal cord. Immunohistochemistry studies confirmed a significant decrease in GFAP-labeled cells at the injury epicenter as well as a decrease in spared tissue after treatment with the antagonists. Functional open field testing revealed no significant locomotor score differences between treated and control animals. Our work is consistent with studies suggesting that astrogliosis is an important event after SCI that limits tissue damage and lesion spreading.


Subject(s)
Gliosis/drug therapy , Purinergic P2 Receptor Antagonists/pharmacology , Pyridoxal Phosphate/analogs & derivatives , Spinal Cord Injuries/drug therapy , Spinal Cord/pathology , Suramin/pharmacology , Animals , Antineoplastic Agents/pharmacology , Astrocytes/drug effects , Astrocytes/metabolism , Behavior, Animal/drug effects , Behavior, Animal/physiology , Female , Glial Fibrillary Acidic Protein/metabolism , Gliosis/pathology , Motor Activity/drug effects , Motor Activity/physiology , Platelet Aggregation Inhibitors/pharmacology , Pyridoxal Phosphate/pharmacology , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Purinergic P2/genetics , Recovery of Function/drug effects , Recovery of Function/physiology , Spinal Cord Injuries/pathology
5.
Dev Neurobiol ; 71(7): 595-607, 2011 Jul.
Article in English | MEDLINE | ID: mdl-20949525

ABSTRACT

Failure of axon regeneration after traumatic spinal cord injury (SCI) is attributable in part to the presence of inhibitory molecular interactions. Recent evidence demonstrates that activation of Eph signaling pathways leads to modulation of growth cone dynamics and repulsion through the activation of ephexin, a novel guanine nucleotide exchange factor (GEF). However, little is known about the expression and modulation of Eph molecular targets in the injured spinal cord. In this study, we determined the expression profile of ephexin after a moderate spinal cord contusion at thoracic level (T10) in young adult rats. Western-blot studies showed increased protein expression in injured rats at 4 and 7 days postinjury (DPI) when compared with control animals. The protein levels returned to normal at 14 DPI and remained steady until 28 DPI. However, immunoprecipitation studies of the phosphorylated ephexin demonstrated that this protein is activated by day 2 until 14 DPI. Expression of ephexin was noticeable in neurons, axons, microglia/macrophages, and reactive astrocytes, and co-localized with EphA3, A4, and A7. These results demonstrate the presence of ephexin in the adult spinal cord and its activation after SCI. Therefore, we show, for the first time, the spatiotemporal pattern of ephexin expression and activation after contusive SCI. Collectively, our data support our previous findings on the putative nonpermissive roles of Eph receptors after SCI and the possible involvement of ephexin in the intracellular cascade of events.


Subject(s)
Guanine Nucleotide Exchange Factors/metabolism , Spinal Cord Injuries/metabolism , Animals , Blotting, Western , Enzyme Activation , Female , Gene Expression , Gene Expression Profiling , Immunohistochemistry , Immunoprecipitation , Rats , Rats, Sprague-Dawley
6.
J Neurotrauma ; 26(10): 1783-93, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19653810

ABSTRACT

Acute pain is a common symptom experienced after spinal cord injury (SCI). The presence of this pain calls for treatment with analgesics, such as buprenorphine. However, there are concerns that the drug may exert other effects besides alleviation of pain. Among those reported are in vitro changes in gene expression, apoptosis, and necrosis. In this investigation, the effect of buprenorphine was assessed at the molecular, behavioral, electrophysiological, and histological levels after SCI. Rats were injured at the T10 thoracic level using the NYU impactor device. Half of the animals received buprenorphine (0.05 mg/kg) for 3 consecutive days immediately after SCI, and the other half were untreated. Microarray analysis (n = 5) was performed and analyzed using the Array Assist software. The genes under study were grouped in four categories according to function: regeneration, apoptosis, second messengers, and nociceptive related genes. Microarray analysis demonstrated no significant difference in gene expression between rats treated with buprenorphine and the control group at 2 and 4 days post-injury (DPI). Experiments performed to determine the effect of buprenorphine at the electrophysiological (tcMMEP), behavioral (BBB, grid walking and beam crossing), and histological (luxol staining) levels revealed no significant difference at 7 and 14 DPI in the return of nerve conduction, functional recovery, or white matter sparing between control and experimental groups (p > 0.05, n = 6). These results show that buprenorphine (0.05 mg/kg) can be used as part of the postoperative care to reduce pain after SCI without affecting behavioral, physiological, or anatomical parameters.


Subject(s)
Analgesics, Opioid/pharmacology , Buprenorphine/pharmacology , Gene Expression Regulation/drug effects , Pain, Intractable/drug therapy , Pain, Intractable/etiology , Recovery of Function/drug effects , Spinal Cord Injuries/complications , Analgesics, Opioid/adverse effects , Animals , Apoptosis/drug effects , Apoptosis/genetics , Buprenorphine/adverse effects , Disease Models, Animal , Evoked Potentials, Motor/drug effects , Evoked Potentials, Motor/physiology , Female , Gait Disorders, Neurologic/chemically induced , Gait Disorders, Neurologic/physiopathology , Gene Expression Regulation/physiology , Locomotion/drug effects , Locomotion/physiology , Nerve Regeneration/drug effects , Nerve Regeneration/genetics , Nerve Tissue Proteins/genetics , Neural Conduction/drug effects , Neural Conduction/physiology , Nociceptors/drug effects , Nociceptors/metabolism , Oligonucleotide Array Sequence Analysis , Pain, Intractable/metabolism , Rats , Rats, Sprague-Dawley , Recovery of Function/physiology , Second Messenger Systems/drug effects , Second Messenger Systems/genetics , Spinal Cord Injuries/metabolism , Spinal Cord Injuries/physiopathology , Treatment Outcome
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