Biomedical subjects

Gregory A Helm

Publications and source records attributed to Gregory A Helm.

40 records · Page 3Linked to original sources

PubMed2002

Caspase-3-mediated cleavage of amyloid precursor protein and formation of amyloid Beta peptide in traumatic axonal injury.

Immunohistochemical studies demonstrate accumulation of the beta-amyloid precursor protein (APP) within injured axons following traumatic brain injury (TBI). Despite such descriptions, little is known about the ultimate fate of accumulating APP at sites of traumatic axonal injury (TAI). Recently, caspase-3-mediated cleavage of APP and subsequent Abeta deposition was linked to apoptotic neuronal death pathways in hippocampal neurons following ischemic and excitotoxic brain injury. Given that (1) APP is known to accumulate within traumatically injured axons, (2) caspase-3 activation has been demonstrated in traumatic axonal injury (TAI), and (3) recent studies have identified a caspase-3 cleavage site within APP, we initiated the current investigation to determine whether caspase-3-mediated cleavage of APP occurs in TAI. We further assessed whether these events were found in relation to Abeta peptide formation. To this end, we employed antibodies targeting APP, the caspase-3-mediated breakdown product of APP proteolysis, and the Abeta peptide. Rats were subjected to impact acceleration TBI (6 h to 10 days survival), and their brains were processed for single-label bright field and multiple double-label immunofluorescent paradigms using the above antibodies. By 12 h postinjury, caspase-3-mediated APP proteolysis (CMAP) was demonstrated within the medial lemniscus (ML) and medial longitudinal fasciculus (MLF) in axons undergoing TAI, identified by their concomitant APP accumulation. Immunoreactivity for CMAP persisted up to 48 h postinjury in the ML and MLF, but was notably reduced by 10 days following injury. Further, CMAP was colocalized with Abeta formation in foci of TAI. The current study demonstrates that caspase-3 cleavage of APP occurs in TAI and is associated with formation of Abeta peptide. These findings are of interest given recent epidemiological studies supporting an association between TBI and later risk for AD development.

Alzheimer Disease↗

PubMed2002

Ex vivo bone morphogenetic protein-9 gene therapy using human mesenchymal stem cells induces spinal fusion in rodents.

OBJECTIVE: Ex vivo gene therapy with the use of human mesenchymal stem cells (hMSCs) and bone morphogenetic protein (BMP) genes provides a local supply of precursor cells and a supraphysiological dose of osteoinductive molecules that may promote bone formation in patients with inadequate hMSC populations because of age, osteoporosis, metastatic bone disease, iatrogenic depletion, or other metabolic derangements. This study was undertaken to evaluate the efficacy of ex vivo gene therapy with the use of hMSCs and the BMP-9 gene to promote spinal fusion in the rat. METHODS: Sixteen athymic nude rats were treated with hMSCs transduced with recombinant, replication-defective Type 5 adenovirus containing the cytomegalovirus promoter and either the BMP-9 (Ad-BMP-9) or the beta-galactosidase (Ad-beta-gal) gene. Ad-beta-gal served as the control. Each animal received a percutaneous, paraspinal injection of 10(6) hMSCs transduced with 50 plaque-forming units/cell adenovirus in the lumbar region, with Ad-BMP-9 on the left and Ad-beta-gal on the right. At 8 weeks postinjection, computed tomographic scans of the lumbosacral spine were obtained, and the lumbosacral spine specimens were examined histologically. RESULTS: Both computed tomographic studies and histological analysis clearly demonstrated large volumes of ectopic bone at the Ad-BMP-9-transduced hMSC injection sites, resulting in successful spinal fusion and no evidence of nerve root compression or local or systemic toxicity. The contralateral regions that were treated with Ad-beta-gal-transduced hMSCs showed no evidence of osteogenesis. CONCLUSION: The results of this study suggest that hMSC and BMP-9 ex vivo gene therapy may be useful in inducing spinal fusion as well as other related procedures and certainly warrants further clinical development.

Animals↗

PubMed2002

Ultrastructural changes of neuronal mitochondria after transient and permanent cerebral ischemia.

BACKGROUND AND PURPOSE: Mitochondrial swelling is one of the most striking and initial ultrastructural changes after acute brain ischemia. The purpose of the present study was to examine the role of reperfusion of the cerebral cortex after transient focal cerebral ischemia on neuronal mitochondrial damage. METHODS: Male Sprague-Dawley rats (n=16) were subjected to either temporary or permanent occlusion of the middle cerebral artery and bilateral carotid arteries. Three experimental conditions were compared: group I, permanent ischemia (3, 5, and 24 hours); group II, transient ischemia (2, 24 hours of reperfusion); and sham surgery. Anesthetized rats were killed by cardiac perfusion, and brain tissue was removed ipsilaterally and contralaterally from the ischemic core section of the frontoparietal cortex. Fixed tissue was prepared for electron microscopic examination, and electron microscopic thin sections of random neurons were photographed. Perinuclear neuronal mitochondria were analyzed in a blinded manner for qualitative ultrastructural changes (compared with sham control) by 2 independent investigators using an objective grading system. RESULTS: Cortical neuronal mitochondria exposed to severe ischemic/reperfusion conditions demonstrated dramatic signs of injury in the form of condensation, increased matrix density, and deposits of electron-dense material followed by disintegration by 24 hours. In contrast, mitochondria exposed to an equivalent time of permanent ischemia demonstrated increasing loss of matrix density with pronounced swelling followed by retention of their shape by 24 hours. CONCLUSIONS: Neuronal mitochondria undergoing transient versus permanent ischemia exhibit significantly different patterns of injury. Structural damage to neuronal mitochondria of the neocortex occurs more acutely and to a greater extent during the reperfusion phase in comparison to ischemic conditions alone. Further research is in progress to delineate the role of oxygen free radical production in the observed mitochondrial damage during postischemic reoxygenation.

Animals↗

PubMed2002

Elastase-induced saccular aneurysms in rabbits: a dose-escalation study.

BACKGROUND AND PURPOSE: Reproducible animal models facilitate preclinical assessment of aneurysm therapies. Our purpose was to determine if increased elastase doses enlarge aneurysms and parent arteries. METHODS: Rabbit right common carotid artery (CCA) aneurysms were created with distal ligation and intraluminal elastase incubation. Groups were 1) sham (no elastase, n = 3), 2) 25% elastase (10 minutes, n = 9), 3) 50% elastase (10 minutes, n = 7), and 4) 50% elastase (20 minutes, n = 41). Angiography was performed after 14 days. Resultant aneurysm width and height and parent artery diameters were measured and compared with the Student t or Mann-Whitney (Wilcoxon rank sum) test. RESULTS: Proximal segments were enlarged in all elastase subjects and no sham subjects. Mean measurements were significantly smaller in the sham group than in other groups. Aneurysm widths and heights, respectively, were 3.8 mm +/- 0.8 and 7.4 mm +/- 2.0 in the low-dose group; 3.9 mm +/- 1.3 and 8.5 mm +/- 3.8, medium-dose group; and 4.1 mm +/- 1.1 and 8.7 mm +/- 2.6, high-dose group. Differences were not significant. Parent artery widths were 3.5 mm +/- 0.7, 3.8 mm +/- 0.7, and 4.3 mm +/- 1.4 in the low-, medium-, and high-dose groups, respectively; the high-dose group had larger arteries (P =.07). CONCLUSION: Aneurysms were reliably created and were sized similar to human intracranial aneurysms. Elastase concentration and incubation duration did not affect resultant size. Relatively short incubation (eg, 10 minutes) and 25% elastase can be used to create rabbit aneurysms, especially if dilatation of adjacent parent arteries is to be avoided.

Angiography, Digital Subtraction↗