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Ronald L Hayes

Publications and source records attributed to Ronald L Hayes.

34 records · Page 2Linked to original sources
PubMed2004

Molecular cloning and characterization of rat and human calpain-5.

Until today, 14 isoforms of mammalian calpains have been identified, including calpain-5. The C. elegans calpain-5 homologue tra-3 is reported to be essential for necrotic neuronal cell death. In this study, we cloned and characterized rat calpain-5, which is highly homologous to human and mouse sequences. The nucleotide sequence is 87% and 93% identical with human and mouse calpain-5, respectively. The protein sequence is well conserved, showing 96% identity in mouse and 92% in human. RT-PCR analysis revealed strong expression of calpain-5 in rat lungs, kidneys, and brain while week expression in heart, whereas in rat brain regions it is ubiquitously expressed. The mRNA expression in different human tissues showed equal expression. However, in human brain regions calpain-5 was strongly expressed in hypothalamus, thalamus, cerebellum, and frontal lobe. Western blot analysis on human neuroblastoma SH-SY5Y cells demonstrated calcium-dependent processing of calpain-5, despite the absence of calmodulin-like domain.

Amino Acid Sequence↗
PubMed2004

Increased expression and processing of caspase-12 after traumatic brain injury in rats.

Traumatic brain injury (TBI) disrupts tissue homeostasis resulting in pathological apoptotic activation. Recently, caspase-12 was reported to be induced and activated by the unfolded protein response following excess endoplasmic reticulum (ER) stress. This study examined rat caspase-12 expression using the controlled cortical impact TBI model. Immunoblots of fractionalized cell lysates found elevated caspase-12 proform (approximately 60 kDa) and processed form (approximately 12 kDa), with peak induction observed within 24 h post-injury in the cortex (418% and 503%, respectively). Hippocampus caspase-12 proform induction peaked at 24 h post-injury (641%), while processed form induction peaked at 6 h (620%). Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis confirmed elevated caspase-12 mRNA levels after TBI. Injury severity (1.0, 1.2 or 1.6 mm compression) was associated with increased caspase-12 mRNA expression, peaking at 5 days in the cortex (657%, 651% and 1259%, respectively) and 6 h in the hippocampus (435%, 451% and 460%, respectively). Immunohistochemical analysis revealed caspase-12 induction in neurons in both the cortex and hippocampus as well as in astrocytes at the contusion site. This is the first report of increased expression of caspase-12 following TBI. Our results suggest that the caspase-12-mediated ER apoptotic pathway may play a role in rat TBI pathology independent of the receptor- or mitochondria-mediated apoptotic pathways.

Animals↗
PubMed2004

Cathepsin B mRNA and protein expression following contusion spinal cord injury in rats.

We provide the first data that cathepsin B (Cath B), a lysosomal cysteine protease, is up-regulated following contusion-spinal cord injury (SCI). Following T12 laminectomy and moderate contusion, Cath B mRNA and protein expression profiles were examined from 2 to 168 h post-injury in rats using real-time PCR and immunoblots, respectively. Contusion injury significantly increased [mRNA]Cath B in the injury site and adjacent segments over sham injury levels. While the largest [mRNA]Cath B induction (20-fold over naive) was seen in the injury site, the caudal segment routinely yielded [mRNA]Cath B levels greater than 10-fold over naive. Interestingly, sham injury animals also experienced mRNA induction at several time points at the injury site and in segments rostral and caudal to the injury site. Contusion injury also significantly elevated levels of Cath B proenzyme protein (37 kDa) over sham injury in the injury site (48, 72 and 168 h post-injury). Furthermore, significant protein increases of single and double chain Cath B (both active forms) occurred at the injury site at 72 and 168 h post-injury. Similar significant increases in Cath B protein levels were seen in areas adjacent to the injury site. The induction of Cath B mRNA and protein expression following contusion injury is previously undescribed and suggests that Cath B may potentially be involved in the secondary injury cascade, perhaps for as long as 1 week post-injury.

Animals↗
PubMed2004

Cell-specific upregulation of survivin after experimental traumatic brain injury in rats.

In this study, we examined the expression and cellular localization of survivin and proliferating cell nuclear antigen (PCNA) after controlled cortical impact traumatic brain injury (TBI) in rats. There was a remarkable and sustained induction of survivin mRNA and protein in the ipsilateral cortex and hippocampus of rats after TBI, peaking at five days post injury. In contrast, both survivin mRNA and protein were virtually undetectable in craniotomy control animals. Concomitantly, expression of PCNA was also significantly enhanced in the ipsilateral cortex and hippocampus of these rats with similar temporal and spatial patterns. Immunohistochemistry revealed that survivin and PCNA were co-expressed in the same cells and had a focal distribution within the injured brain. Further analysis revealed a frequent co-localization of survivin and GFAP, an astrocytic marker, in both the ipsilateral cortex and hippocampus, while a much smaller subset of cells showed co-localization of survivin and NeuN, a mature neuronal marker. Neuronal localization of survivin was observed predominantly in the ipsilateral cortex and contralateral hippocampus after TBI. PCNA protein expression was detected in both astrocytes and neurons of the ipsilateral cortex and hippocampus after TBI. Collectively these data demonstrate that the anti-apoptotic protein survivin, previously characterized in cancer cells, is abundantly expressed in brain tissues of adult rats subjected to TBI. We found survivin expression in both astrocytes and a sub-set of neurons. In addition, the expression of survivin was co-incident with PCNA, a cell cycle protein. This suggests that survivin may be involved in regulation of neural cell proliferative responses after traumatic brain injury.

Animals↗
PubMed2004

Lysophosphatidic acid induces clonal generation of mouse neurospheres via proliferation of Sca-1- and AC133-positive neural progenitors.

Neural stem/progenitor cells are clonogenic in vitro and produce neurospheres in serum-free medium containing epidermal growth factor (EGF) and fibroblast growth factor (FGF2). Here, we demonstrate that lysophosphatidic acid (LPA) instigated the clonal generation of neurospheres from dissociated mouse postnatal forebrain in the absence of EGF and FGF2. LPA induced proliferation of cells which co-expressed Sca-1 antigen and AC133, markers of primitive hematopoietic and neural stem/progenitor cells. Clonal expansion of these cells induced by LPA was inhibited by diacylglycerol- pyrophosphate (DGPP), an antagonist of the LPA receptor subtypes LPA1 and LPA3. Moreover, Sca-1- and AC133-positive cells of these neurospheres expressed LPA1, LPA2, and LPA3, suggesting important roles for these LPA receptors in proliferation of neural progenitors. LPA induced neurospheres to differentiate on an adherent laminin/poly-L-ornithine matrix. In differentiating neurospheres, LPA receptors co-localized with betaIII-tubulin, nestin, and CNPase, but not with glial fibrillary acidic protein (GFAP), a marker of astrocyte lineage. Our results demonstrate for the first time that lysophosphatidic acid induces clonal neurosphere development via proliferation of AC133/Sca-1-positive stem cells by a receptor-dependent mechanism. This differentiation was characterized by the initial co-localization of neural specific antigens at sites of LPA receptor expression upon their interaction with the inducing agonist.

AC133 Antigen↗
PubMed2004

Accumulation of calpain and caspase-3 proteolytic fragments of brain-derived alphaII-spectrin in cerebral spinal fluid after middle cerebral artery occlusion in rats.

Preclinical studies have identified numerous neuroprotective drugs that attenuate brain damage and improve functional outcome after cerebral ischemia. Despite this success in animal models, neuroprotective therapies in the clinical setting have been unsuccessful. Identification of biochemical markers common to preclinical and clinical cerebral ischemia will provide a more sensitive and objective measure of injury severity and outcome to facilitate clinical management and treatment. However, there are currently no effective biomarkers available for assessment of stroke. Nonerythroid alphaII-spectrin is a cytoskeletal protein that is cleaved by calpain and caspase-3 proteases to signature alphaII-spectrin breakdown products (alphaII-SBDPs) after cerebral ischemia in rodents. This investigation examined accumulation of calpain- and caspase-3-cleaved alphaII-SBDPs in cerebrospinal fluid (CSF) of rodents subjected to 2 hours of transient focal cerebral ischemia produced by middle cerebral artery occlusion (MCAO) followed by reperfusion. After MCAO injury, full-length alphaII-spectrin protein was decreased in brain tissue and increased in CSF from 24 to 72 hours after injury. Whereas alphaII-SBDPs were undetectable in sham-injured control animals, calpain but not caspase-3 specific alphaII-SBDPs were significantly increased in CSF after injury. However, caspase-3 alphaII-SBDPS were observed in CSF of some injured animals. These results indicate that alphaII-SBDPs detected in CSF after injury, particularly those mediated by calpain, may be useful diagnostic indicators of cerebral infarction that can provide important information about specific neurochemical events that have occurred in the brain after acute stroke.

Animals↗
PubMed2004

Increased expression of tissue-type transglutaminase following middle cerebral artery occlusion in rats.

Tissue-type transglutaminase (TG-2) has been implicated in neurodegenerative diseases. In this study, induction of TG-2 was studied in rats following transient middle cerebral artery occlusion. Alterations in 2,3,5-triphenylterazolium chloride staining revealed maximum infarction 3 days after injury. Measurement of mRNA transcript levels by real-time PCR analysis showed both forms of TG-2 mRNA peaking on day 5 after injury in ipsilateral cortex, with greater induction of the full-length TG-2 (TG-L) transcript than the truncated form of the TG-2 (TG-S) transcript. However, in the ipsilateral hippocampus, peak induction of both forms of TG-2 mRNA peaked 1 day after injury and to a lesser extent than observed in the ipsilateral cortex. Western blot analysis demonstrated that TG-L protein expression progressively increased from 1 to 7 days after ischemia, with greater expression in cortex than hippocampus (525 +/- 10% vs. 196 +/- 8% of control, respectively). However, expression of TG-S was not detected. These results demonstrate that increased TG-2 mRNA and protein expression occurs in a delayed fashion following ischemic injury. The temporal profile of TG-2 induction after ischemia was similar to that observed after traumatic brain injury (previously described), suggesting a similar role of TG-2 in both pathological conditions.

Animals↗
PubMed2004

Biomarkers of proteolytic damage following traumatic brain injury.

The history of numerous failed clinical trials designed to identify therapeutic agents to assist in improving outcomes after traumatic brain injury points to the critical importance of understanding biochemical markers of injury. Such biomarkers should be readily accessible, provide information specific to the pathologic disruptions occurring in the central nervous system, and allow improved monitoring of the progression of secondary damage. Additionally, these biomarkers should may provide investigators a window on the individual patient's response to treatment, and should contribute to prediction of outcome. Most research on this topic to date has focused on neuronspecific enolase (NSE) and S-100 proteins but these have not proven to be satisfactory for a variety of reasons. A different approach is provided by the study of 2 important proteases, caspase-3 and calpain. This paper reports the current state of knowledge concerning caspase and calpain as specific markers of TBI, and discusses all-spectrin, a principal substrate for both caspase and calpain, as well as initial findings regarding neurofilament 68 protein (NF-68).

Animals↗
PubMed2003

Application of proteomics technology to the field of neurotrauma.

Near-completion of the Human Genome Project has stimulated scientists to begin looking for the next step in unraveling normal and abnormal functions within biological systems. Consequently, there is new focus on the role of proteins in these processes. Proteomics is a burgeoning field that may provide a valuable approach to evaluate the post-traumatic central nervous system (CNS). Although we cannot provide a comprehensive assessment of all methods for protein analysis, this report summarizes some of the newer proteomic technologies that have propelled this field into the limelight and that are available to most researchers in neurotrauma. Three technical approaches (two-dimensional gel electrophoresis, direct analysis by mass spectrometry, including two-dimensional chromatography coupled to mass spectrometry and isotope coded affinity tags, and antibody technologies) are reviewed, and their advantages and disadvantages presented. A discussion of proteomic technology in the context of brain and spinal cord trauma follows, addressing current and future challenges. Proteomics will likely be very useful for developing diagnostic predictors after CNS injury and for mapping changes in proteins after injury in order to identify new therapeutic targets. Neurotrauma results in complex alterations to the biological systems within the nervous system, and these changes evolve over time. Exploration of the "new nervous system" that follows injury will require methods that can both fully assess and simplify this complexity.

Antibodies↗
PubMed2003

Diffusion magnetic resonance imaging study of a rat hippocampal slice model for acute brain injury.

Diffusion magnetic resonance imaging (MRI) provides a surrogate marker of acute brain pathology, yet few studies have resolved the evolution of water diffusion changes during the first 8 hours after acute injury, a critical period for therapeutic intervention. To characterize this early period, this study used a 17.6-T wide-bore magnet to measure multicomponent water diffusion at high b-values (7 to 8,080 s/mm(2)) for rat hippocampal slices at baseline and serially for 8 hours after treatment with the calcium ionophore A23187. The mean fast diffusing water fraction (Ffast) progressively decreased for slices treated with 10-microM/L A23187 (-20.9 +/- 6.3% at 8 hours). Slices treated with 50-micromol/L A23187 had significantly reduced Ffast 80 minutes earlier than slices treated with 10-microM/L A23187 (P < 0.05), but otherwise, the two doses had equivalent effects on the diffusion properties of tissue water. Correlative histologic analysis showed dose-related selective vulnerability of hippocampal pyramidal neurons (CA1 > CA3) to pathologic swelling induced by A23187, confirming that particular intravoxel cell populations may contribute disproportionately to water diffusion changes observed by MRI after acute brain injury. These data suggest diffusion-weighted images at high b-values and the diffusion parameter Ffast may be highly sensitive correlates of cell swelling in nervous issue after acute injury.

Acute Disease↗
PubMed2002

Up-regulation of tissue-type transglutaminase after traumatic brain injury.

Tissue-type transglutaminase (tTG, EC 2.3.2.13) has been implicated in various disease paradigms including neurodegenerative disease. In these studies, tTG induction after traumatic brain injury was studied using a rat cortical impact model. Using western blots, two forms of tTG protein expression were identified--an approximately 79-kDa primary form (tTG-L) and a less abundant approximately 70-kDa form (tTG-S). Both forms of tTG protein were elevated after injury. In ipsilateral cortex, peak induction of tTG-L protein [561% +/- 80% of control (n = 5)] was observed five days after injury, with expression remaining elevated after two weeks. Peak induction of tTG-S protein [302% +/- 81% of control (n = 5)] was observed three days after injury. Lesser tTG protein induction was observed in hippocampus. Northern blot analysis demonstrated two tTG transcripts in the ipsilateral cortex with peak induction of tTG-L mRNA three days after injury. However, tTG-S mRNA was not identified in control samples and only faintly detected in injured tissue. To facilitate analysis of low abundance transcripts in smaller tissue samples, a semiquantitative real-time PCR strategy was used. Semi-quantitative PCR analysis of tTG-L mRNA induction in ipsilateral cortex (peak after three days; 414% +/- 21% of control, n = 3) confirmed tTG-L mRNA induction determined by northern blot (410% of control).

Animals↗
PubMed2002

Temporal and spatial profile of Bid cleavage after experimental traumatic brain injury.

Apoptosis plays an essential role in the cascade of CNS cell degeneration after traumatic brain injury. However, the underlying mechanisms are poorly understood. The authors examined the temporal profile and cell subtype distribution of the proapoptotic protein Bid from 6 hours to 7 days after cortical impact injury in the rat. Increased protein levels of tBid were seen in the cortex ipsilateral to the injury site from 6 hours to 3 days after trauma. Immunohistologic examinations revealed expression of tBid in neurons, astrocytes, and oligodendrocytes from 6 hours to 3 days after impact injury, and concurrent assessment of DNA damage using TUNEL identified tBid-immunopositive cells with apoptoticlike morphology in the traumatized cortex. Moreover, Bid cleavage and activation of caspase-8 and caspase-9 occurred at similar time points and in similar brain regions (i.e., cortical layers 2 to 5) after impact injury. In contrast, there was no evidence of caspase-8 or caspase-9 processing or Bid cleavage in the ipsilateral hippocampus, contralateral cortex, and hippocampus up to 7 days after the injury. The results provide the first evidence of Bid cleavage in the traumatized cortex after experimental traumatic brain injury in vivo, and demonstrate that tBid is expressed in neurons and glial cells. Further, findings indicate that cleavage of Bid may be associated with the activation of the initiator caspase-8 and caspase-9. Finally, these data support the hypothesis that cleavage of Bid contributes to the apoptotic degeneration of different CNS cells in the injured cortex.

Animals↗
PubMed1992

Pretreatment with gangliosides reduces abnormal nociceptive responses associated with a rodent peripheral mononeuropathy.

A peripheral mononeuropathy was produced in adult male rats by placing loosely constrictive ligatures around the common sciatic nerve. As reported by others, this procedure reliably results in postoperative behavior indicative of hyperalgesia, allodynia, and potentially, spontaneous pain. In these experiments, thermal hyperalgesia was assessed by measuring foot-withdrawal latencies to radiant heat aimed at the plantar surface of rat hind paws. Behaviors potentially indicative of spontaneous pain were assessed by rating spontaneous hind paw guarding positions. Rats with sciatic nerve ligation were divided into 5 groups (n = 6/group). Three groups received injections (i.p.) of either 10, 20 or 40 mg/kg of cerebral ganglioside mixture, GA. The 4th group was injected with 10 mg/kg of the purified ganglioside GM1, and the 5th group received an equal volume of saline. All injections were given daily for 2 days before surgery, the day of surgery and 9 days after surgery. All animals were behaviorally assessed for 2 days prior to surgery, the day of surgery, as well as 1, 3, 5, 7, and 10 days afterwards. All 5 groups had significantly reduced latencies to hind paw withdrawal on the side ipsilateral to sciatic nerve ligation. However, these hyperalgesic responses were significantly attenuated in rats receiving GA or GM1 pretreatment. These data suggest that this animal model of peripheral neuropathic pain is sensitive to pharmacological manipulations useful for understanding mechanisms of neuropathic pain, including mechanisms related to excitotoxic processes. Such studies could lead to development of clinical approaches to treat this disorder.

Animals↗
PubMed1992

Spatial patterns of spinal cord [14C]-2-deoxyglucose metabolic activity in a rat model of painful peripheral mononeuropathy.

Spatial patterns of spinal cord glucose metabolic activity were examined in unanesthetized rats with painful peripheral mononeuropathy produced by sciatic nerve ligation (chronic constrictive injury, CCI). Spinal cord metabolic activity was assessed 10 days after nerve ligation by using the fully quantitative [14C]2-deoxyglucose technique. This technique allows simultaneous examination of both neural activity inferred from local glucose utilization and its spatial distribution in multiple spinal regions previously implicated in nociceptive processing. Rats used in the experiment exhibited thermal hyperalgesia to radiant heat applied to the hind paw ipsilateral to nerve ligation and behaviors indicative of spontaneous pain. Sciatic nerve ligation produced a significant increase in spinal cord metabolic activity in four sampling regions (laminae I-IV, V-VI, VII and VIII-IX) of lumbar segments compared to sham-operated rats. The pattern of altered metabolic activity in CCI rats presented 3 distinct features. (1) The spinal cord grey matter both ipsilateral and contralateral to nerve ligation exhibited substantial increases in metabolic activity compared to sham-operated rats. (2) This increase in metabolic activity was somatotopically specific, i.e., higher metabolic rates were observed on the side ipsilateral to nerve ligation than on the contralateral side, and higher metabolic rates were seen in the medial portion of the ipsilateral spinal cord dorsal horn than in the lateral portion. The peak metabolic activity occurred in laminae V-VI of CCI rats, a region involved in nociceptive processing. (3) The increase in spinal cord metabolic activity of CCI rats extended from lumbar segment L1 to L5 in all 4 sampling regions. The substantial increase in metabolic activity in both the ipsilateral and contralateral spinal cord that occurs over an extensive rostro-caudal area in CCI rats may represent a unique pattern of spinal cord metabolic activity distinct from that observed in rats exposed to acute thermal pain. This pattern of spinal cord neural activity in CCI rats may reflect possible radiation of neuropathic pain. In addition, the procedure of curare-induced paralysis in a separate group of CCI rats did not change the extent and patterns of metabolic activity seen in non-paralyzed CCI rats, reflecting a minimal influence of the afferent feedback from flexor motor reflexes on spinal cord metabolic activity following sciatic nerve ligation. This chronic increase in spinal cord neural activity in the absence of overt peripheral stimulation suggests a spinal cord hyperactive state and may account for behaviors suggestive of spontaneous pain in CCI rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMedDate not supplied

Neuroproteomics in neurotrauma.

Neurotrauma in the form of traumatic brain injury (TBI) afflicts more Americans annually than Alzheimer's and Parkinson's disease combined, yet few researchers have used neuroproteomics to investigate the underlying complex molecular events that exacerbate TBI. Discussed in this review is the methodology needed to explore the neurotrauma proteome-from the types of samples used to the mass spectrometry identification and quantification techniques available. This neuroproteomics survey presents a framework for large-scale protein research in neurotrauma, as applied for immediate TBI biomarker discovery and the far-reaching systems biology understanding of how the brain responds to trauma. Ultimately, knowledge attained through neuroproteomics could lead to clinical diagnostics and therapeutics to lessen the burden of neurotrauma on society.

Animals↗