Initial Ischemic Injury Research Articles

Novel Vascular Lesions in Mice Given a Non-Peptide Vitronectin Receptor Antagonist

Novel vascular lesions were observed in mice given an alpha vbeta 3, alpha vbeta 5 receptor antagonist (SB-273005) for up to 3 months. Vascular smooth muscle cell (VSMC) necrosis was observed in aorta and renal hilar arteries approximately 6 hours after dosing followed by loss of VSMC, adaptive medial thickening by VSMC hypertrophy and deposition of PAS-positive matrix and collagen. Renal hilar and arcuate arteries developed delayed and transient fibrinoid necrosis and inflammation. Vascular regeneration was not evident following drug-withdrawal after 3 days of dosing. Vascular lesions were associated with necrosis, regeneration and fibrosis of heart, kidney and spleen consistent with initial ischemic injury followed by tissue repair. VSMC toxicity was likely not related to integrin antagonism because lesions were not observed with related compounds and no vascular changes were observed in other preclinical species. In vitro studies failed to demonstrate a direct toxic effect of SB-273005 on VSMC or unique species sensitivity of murine VSMC. In conclusion, SB-273005 caused VSMC necrosis in aorta and renal arteries of mice. Lesions did not progress or recover, but there was medial hypertrophic adaptation even with continued dosing. This is considered direct species-specific VSMC toxicity of unknown mechanism and unrelated to vitronectin receptor antagonism.

Infarct Prediction and Treatment Assessment with MRI-based Algorithms in Experimental Stroke Models

There is increasing interest in using algorithms combining multiple magnetic resonance imaging (MRI) modalities to predict tissue infarction in acute human stroke. We developed and tested a voxel-based generalized linear model (GLM) algorithm to predict tissue infarction in an animal stroke model in order to directly compare predicted outcome with the tissue's histologic outcome, and to evaluate the potential for assessing therapeutic efficacy using these multiparametric algorithms. With acute MRI acquired after unilateral embolic stroke in rats (n=8), a GLM was developed and used to predict infarction on a voxel-wise basis for saline (n=6) and recombinant tissue plasminogen activator (rt-PA) treatment (n=7) arms of a trial of delayed thrombolytic therapy in rats. Pretreatment predicted outcome compared with post-treatment histology was highly accurate in saline-treated rats (0.92+/-0.05). Accuracy was significantly reduced (P=0.04) in rt-PA-treated animals (0.86+/-0.08), although no significant difference was detected when comparing histologic lesion volumes. Animals that reperfused had significantly lower (P<0.01) GLM-predicted infarction risk (0.73+/-0.03) than nonreperfused animals (0.81+/-0.05), possibly reflecting less severe initial ischemic injury and therefore tissue likely more amenable to therapy. Our results show that acute MRI-based algorithms can predict tissue infarction with high accuracy in animals not receiving thrombolytic therapy. Furthermore, alterations in disease progression due to treatment were more sensitively monitored with our voxel-based analysis techniques than with volumetric approaches. Our study shows that predictive algorithms are promising metrics for diagnosis, prognosis and therapeutic evaluation after acute stroke that can translate readily from preclinical to clinical settings.

Matrix metalloproteinase induction in post-transplant obliterative bronchiolitis

Background Epithelial cell injury, inflammation, fibrosis, and airway obliteration are associated in post-transplant obliterative bronchiolitis. Fibrosis is a consequence of fibroblastic activity and of collagen deposition after disturbances in the balance of protein formation and degradation. Proteolytic enzymes such as the matrix metalloproteinases mediate degradation. To assess matrix metalloproteinases during obliterative bronchiolitis development, we studied porcine, heterotopic bronchial allografts. Methods A total of 119 allografts or autografts were harvested serially at 3 to 60 days after transplantation and processed for histology and in situ hybridization for matrix metalloproteinases 2 and 9. Immunocytochemistry for vimentin and α-smooth-muscle-cell actin was performed with specific antibodies. Results Implants had initial ischemic injury to airway epithelium and to the bronchial wall. Recovery was rapid in autografts and in immunosuppressed allografts. In matrix metalloproteinase-2 mRNA activity in fibroblasts, correlation with endothelial expression and expression in macrophages occurred during intense fibroproliferation. We observed intense matrix metalloproteinase-9 positivity during onset of inflammation and fibroproliferation in endothelial cells ( p < 0.01), fibroblasts ( p < 0.05), macrophages ( p < 0.05), and lymphocytes ( p < 0.05). Matrix metalloproteinase-9 mRNA activity in fibroblasts correlated with that in endothelial and inflammatory cells and also proved predictive of early obliteration. Conclusions Matrix metalloproteinase-2, and especially matrix metalloproteinase-9, gene activity was associated with onset of inflammation and fibroblastic proliferation in allografts, predicting early obliteration. Although this may be the case in the model described, its role in human-allograft post-transplant obliterative bronchiolitis requires further supportive data.

Respiratory epithelial expression of integrin αvβ6 in chronic progressive allograft rejection

Background Obliterative bronchiolitis (OB) is the major cause of morbidity and mortality after lung transplantation. One initiating event in the development of obliteration of the airway lumen is epithelial injury. In our model of chronic rejection, initial ischemic injury and denudation of the epithelium occurs in the isografts, with eventual re-epithelialization and partial patency of the airway lumen. In contrast, allografts do not recover epithelium, and the airway lumen becomes obliterated. We hypothesized that because integrin αVβ6 is expressed in healing epithelium, integrin αVβ6 expression would be greatly increased in isografts, but not in allografts. Methods Using a rat tracheal allograft rejection model as a source of 4- to 5-μm tissue sections, we compared integrin staining in allografts vs isografts from animals at post-transplant Days 7, 14, 28, and 60. We analyzed the sections using immunohistochemistry after incubation with a specific monoclonal antibody E7P6 against integrin αVβ6. Negative control slides were processed identically, except that primary antibody was omitted. Results The sections from healing, re-epithelializing isografts showed intense staining when using the antibody recognizing integrin αVβ6, compared with the allografts studied. Days 7 and 14 isografts had increased epithelial expression of αVβ6. As the isograft epithelium recovered, the intensity diminished at Days 28 and 60. In allografts, at Days 7 to 60, we detected only a comparatively low-level of expression in injured epithelium. Conclusions Integrin αVβ6 is readily detectable in healing isografts. Integrin αVβ6 may be crucial in maintaining a viable epithelial cell layer, which is related to slowed progression of airway obliteration in OB.

The role of diffusion tensor imaging in the evaluation of ischemic brain injury - a review.

Water diffusion in brain tissue is affected by the presence of barriers to translational motion such as cell membranes and myelin fibers. The measured water apparent diffusion coefficient (ADC) value is therefore frequently anisotropic and varies depending upon the orientation of restricting barriers (such as white matter tracts) relative to the diffusion-sensitive-gradient direction. Anisotropic water diffusion can be specified using indices of diffusion anisotropy [e.g. standard deviation of the individual ADC values, fractional anisotropy (FA), lattice index (LI)], which are derived from measurements of the full diffusion tensor. The rotationally invariant nature of particular diffusion anisotropy indices (e.g. FA, LI) allows orientation-independent comparisons of these parameters between different subjects. Pathophysiological processes (such as cerebral ischemia) that modify the integrity of the tissue microstructure result in significant alterations in tissue anisotropy and make this metric a useful endpoint for characterizing the temporal evolution of the disease. Diffusion-tensor imaging (DTI) studies of both experimental and human stroke suggest that DTI may provide additional information about the evolution of the disease that is not available from diffusion-weighted MRI (DWI) alone. Acute reductions in the average diffusivity [<D> = (lambda(1) + lambda(2) + lambda(3))/3 where lambda(1), lambda(2), and lambda(3) are the eigenvalues of the diffusion tensor] following the onset of cerebral ischemia are often accompanied by increases in diffusion anisotropy. In the transition from acute to sub-acute and chronic stroke, <D> renormalizes and subsequently increases whereas diffusion anisotropy measures (e.g. FA) decline and remained reduced in chronic infarcts. Overall isotropic ADC changes during infarct evolution have been observed to be greater in white matter (WM) than in gray matter (GM) lesions (although there have been conflicting reports on this issue) and GM lesions tend to renormalize prior to WM lesions as the infarct evolves. Ischemic WM exhibits a significant decrease in diffusion anisotropy (relative to normal WM) during ischemic evolution whereas that of ischemic GM remains statistically unchanged. Furthermore, the percentage decrease in ischemic WM <D> is largely determined by reductions in lambda(1), the eigenvalue that coincides with the long axis of the WM fiber tract. Variations in unidirectional ADC or <D> over the ischemic time course limit the usefulness of this parameter alone as a predictor of ischemic injury. Consequently, ADC information has been combined with that of other MR parameters (including DTI) to unambiguously stage and predict ischemic brain injury over its entire temporal evolution. Combined <D> and diffusion anisotropy measurements have identified three phases of diffusion abnormality: (1) reduced <D> and elevated anisotropy; (2) reduced <D> and reduced anisotropy; and (3) elevated <D> and reduced anisotropy. However, variations in the differential patterns of <D> and diffusion anisotropy evolution have been observed by a number of investigators and more work is needed to clarify the role of these measurements in characterizing the severity of the ischemic insult as well as the potential outcome in response to the initial ischemic injury. The use of DTI, in combination with more sophisticated analysis methods for performing multiparametric segmentation, such as multispectral analysis, may enhance the use of MRI for accurate diagnosis and prognosis of stroke. Furthermore, these techniques may also play an important role in the clinical evaluation of new stroke treatments.

Alloimmune injury preceding airway obliteration in porcine heterotopic lung implants: a histologic and immunohistologic study.

Obliterative bronchiolitis (OB), the major long-term complication of lung transplantation, has thus far lacked a good large-animal model. Our goal was to develop such a model on the basis of previous rodent models with tracheal implants. Fragments of pulmonary tissue with structures of terminal bronchi were subcutaneously transplanted to four random-bred domestic piglets. Each animal received 10 autograft and 10 allograft implants. The histologic findings were graded from 0 to 3 for implants harvested repeatedly over 2 months. In autografts, partial destruction of the respiratory epithelium and gradual luminal obliteration as well as mild damage to the cartilage and the bronchial wall underwent rapid reversal after initial ischemic injury. In the allografts, epithelial destruction and gradual obliteration were total within 14 days, the difference being statistically significant (P<0.05) in both. The histologic features of the obliterative plug were similar to those of human OB. In the allografts, cartilaginous destruction and pericartilaginous inflammation increased gradually to severe levels, significantly worse than in the autografts (P<0.05). Necrosis and inflammation of the bronchial wall were also more severe in the allografts (P<0.05). At the end of follow-up, all autografts were vital, whereas the allografts were almost totally rejected and were without native structures. All bronchi in the allografts exhibited accelerated obliteration with histologic features characteristic of human OB, thus providing a model for research into OB and its prevention.

Current understanding of chemokine involvement in allograft transplantation.

Multiple studies have demonstrated that chemokines play an essential role in regulating and co-ordinating the infiltration of leucocytes into allografts. Chemokines are expressed in skin, liver, heart, and kidney allografts following initial engraftment, ischemic injury, viral infection, and acute and chronic rejection. To date, most of our understanding of chemokine biology has been generated from studies of animal models of transplantation and little is known about the role of chemokines in human allograft rejection. Chemokines may play important mechanistic roles in transplant rejection, in the development of graft arteriosclerosis, and in chronic sclerosing cholangiopathy. Furthermore, these molecules may serve as sensitive diagnostic indicators for the analysis of rejection, including chronic rejection or other forms of graft dysfunction. Lastly, it is possible that chemokine-targeted therapy might become a feasible option for the treatment of allograft rejection.

Magnesium and cardiovascular disease.

Magnesium and cardiovascular disease.

Transient forebrain ischemia induces delayed injury in the substantia nigra reticulata: degeneration of GABA neurons, compensatory expression of GAD mRNA

In rodents, transient forebrain ischemia causes preferentially neuron death in small and medium size neurons of the striatum and hilar neurons in the hippocampus within 24 h, and CA1 hippocampal neurons within 72 h. The temporal unfolding of pathological processes after longer time intervals between reperfusion and sacrifice now includes delayed degeneration of the substantia nigra reticulata (SNr). Animals were exposed to 20 min of transient forebrain ischemia and sacrificed within 7 days, or at least 3 weeks after reperfusion. Histological examination and quantitative morphometrics revealed that the degree of volume loss and neuron loss in the SNr depended on the initial ischemic injury. Initial ischemic injury confined to the caudate nucleus produced volume loss but not neuron loss in the SNr. However, initial ischemic injury that included the caudate nucleus and the globus pallidus produced not only greater volume loss but also neuron loss in the SNr. SNr neuron loss was restricted to the medial dorsal area, occurred in animals that survived at least 3 weeks after perfusion, and did not occur in animals that survived 7 days after perfusion, and was accompanied by increased staining of antibody to glial fibrillary acidic protein. The topographic specificity and delayed time course suggest that the mechanism for SNr neuron loss depends on transneuronal events initiated by ischemia but evolving over a longer time period. In situ hybridization with a cDNA probe for glutamic acid decar☐ylase (GAD) mRNA demonstrated increased GAD signal in the remaining SNr neurons of animals with CN and GP damage compared to animals with CN damage. The significant increase in GAD mRNA may indicate compensation at the level of gene expression for the loss of GABAergic neurons. This rodent model offers new in vivo opportunities to elucidate the requirements for neuronal viability, and phenotypic expression, and suggests that the current notions of windows of opportunity for therapeutic intervention may be expanded from hours to days to weeks.

Value and Limitations of Precordial ST-Segment Mapping

The importance of noninvasive techniques for following the evolution of ischemic injury in patients with acute myocardial infarction is indisputable. The current great interest in such methods stems from the realization that damage to more than 40% of left ventricular myocardium is incompatible with survival,¹and from extrapolation to the clinical situation of experimental data, which suggests that the initial ischemic injury may require some period of time for demarcation and cell death to occur.² One technique for following the progression of ischemic injury is the method of serial precordial mapping of the ECG. Clinical application of precordial mapping has followed several years of experimental work, during which epicardial electrograms were correlated with histological and histochemical evidence of necrosis resulting from coronary occlusion.³The observation that ST-segment elevations recorded simultaneously from the epicardium and precordium in dogs move in parallel during experimental manipulations that alter the magnitude

Observations on factors affecting local forces in the left ventricular wall during acute myocardial ischemia.

The factors affecting local myocardial function in the presence of ischemic myocardial damage may considerably influence cardiac function and the extent of tissue damage resulting from the initial ischemic injury. However, previous attempts to study local wall forces in the ischemic myocardium have produced variable results. In the present study, a technique was developed for recording isometric left ventricular wall tension that minimized artifacts due to external forces acting on the ischemic area. Miniature isometric force gauges were implanted in 23 dogs in the left ventricular wall within a zone buffered by two rows of transmural prongs attached to a ring. During a test occlusion of a branch of the left anterior descending coronary artery, the ischemic area was defined by the presence of epicardial ST segment elevation, and force gauges were implanted in marginal or ischemic areas of the myocardium. During a subsequent coronary occlusion wall force in the ischemic area fell immediately by an average of 31 ± 8%. When the occlusion was repeated during isoproterenol infusion, wall tension decreased in previously unaffected areas of the myocardium, although in ischemic areas it remained above previous control ischemic levels. Arterial counterpulsation improved the development of wall tension in the ischemic area by 27% and decreased the extent and amount of ST segment elevation. Thus the present study clearly indicates a significant reduction but not elimination of active myocardial wall force in ischemic zones following experimental coronary occlusion. Moreover, force could be augmented by either isoproterenol or counterpulsation, and counterpulsation can produce a significant improvement in function of the ischemic myocardium while reducing the area of ischemic injury.

Ultrastructural features of canine hepatic auxiliary transplant rejection

Auxiliary canine hepatic homografts were examined electron microscopically 24 hours to 17 days after transplantation. Many nonspecific changes were present. Early, the endoplasmic reticulum was dilated and the mitochondria were abnormal. After the initial postoperative period, autophagic vacuoles, shedding of cytoplasm, light and dark cells, and acidophilic bodies were present and macrophages were common. Initial ischemic cell injury can be explained by vascular derangements inherent in the process of transplantation. Its persistency may result from impaired perfusion by excess mesenchymal cells in sinusoids and tissue spaces and by competition between these and hepatocytes for nutrients. An immunologic component is suggested by the presence of large mononuclear cells, with many ribosomes seen in delayed type hypersensitivity reactions. These were closely approximated to and indenting the hepatic parenchymal cells, but bridging between the mesenchymal cells or hepatocytes was not found. The similarity of the mesenchymal cells and their relation to hepatocytes in chronic active hepatitis in man supports the idea that in both transplant rejection and in the human disease an immunologic process mediated by cells adds to the destruction of hepatic tissue.