Non-uniform upregulation of the autogenic stretch reflex among hindlimb extensors following lateral spinal lesion in the cat.

Successful propagation throughout the step cycle is contingent on adequate regulation of whole-limb stiffness by proprioceptive feedback. Following spinal cord injury (SCI), there are changes in the strength and organization of proprioceptive feedback that can result in altered joint stiffness. In this study, we measured changes in autogenic feedback of five hindlimb extensor muscles following chronic low thoracic lateral hemisection (LSH) in decerebrate cats. We present three features of the autogenic stretch reflex obtained using a mechanographic method. Stiffness was a measure of the resistance to stretch during the length change. The dynamic index documented the extent of adaptation or increase of the force response during the hold phase, and the impulse measured the integral of the response from initiation of a stretch to the return to the initial length. The changes took the form of variable and transient increases in the stiffness of vastus (VASTI) group, soleus (SOL), and flexor hallucis longus (FHL), and either increased (VASTI) or decreased adaptation (GAS and PLANT). The stiffness of the gastrocnemius group (GAS) was also variable over time but remained elevated at the final time point. An unexpected finding was that these effects were observed bilaterally. Potential reasons for this finding and possible sources of increased excitability to this muscle group are discussed.

Apoptotic DNA fragmentation in the rat cerebral cortex induced by permanent middle cerebral artery occlusion.

Recent investigations have demonstrated internucleosomal DNA fragmentation in ischemic neuronal tissue. This type of fragmentation is characteristic of programmed cell death or apoptosis and suggests that neuronal death in stroke may be more complex than simple necrotic death. The present experiments provide a detailed examination of the regional localization and time course for apoptotic DNA fragmentation in the cerebral cortex following focal cerebral ischemia. Spontaneously hypertensive rats were subjected to permanent right middle cerebral artery occlusion and the cerebral cortices were examined for evidence of DNA fragmentation using electrophoretic, flow cytometric, and histological approaches. An electrophoretic examination of cortical DNA at 24 h after the occlusion indicated that the majority of nucleosomal ladders were in the transition zone or penumbra and the core of the infarction, with no fragmentation apparent in the contralateral normal cortex. A flow cytometric analysis of DNA fragmentation in intact cells revealed a similar pattern, with increased fragmentation observed in ischemic cortex vs. the contralateral cortex. Saggital sections taken 1.5 mm lateral to midline were collected from animals at 1, 4, and 24 h after the infarction and DNA fragmentation was examined histologically by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) staining. Quantitative analysis of these sections indicated that DNA fragmentation can be observed in the anterior and central area of the infarctions as soon as 1 h after the occlusion and that the extent and magnitude of the fragmentation increases at 4 and 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased vascular endothelial growth factor expression associated with tumor regression induced by (E)-2'-deoxy-2'-(fluoromethylene)cytidine (MDL 101,731).

The effect of the antitumor drug MDL 101,731 [(E)-2'-deoxy-2'-(fluoromethylene)cytidine] on tumor growth and on steady-state vascular endothelial growth factor (VEGF) mRNA levels in MDA-MB-231, PC-3, MCF-7, and HT-29 human tumor xenografts grown in nude mice was examined, using quantitative in situ hybridization. MDL 101,731 caused regression of MDA-MB-231 and PC-3 tumor xenografts, but only inhibition of growth (without regression) of MCF-7 xenografts. The drug caused inhibition of growth of HT-29 xenografts at low doses, and regression at high doses. When treatment with MDL 101,731 led to tumor regression, VEGF mRNA levels were decreased. When treatment led only to inhibition of growth, there was no significant change in VEGF mRNA. Further examination of the tumor xenografts revealed that elevated VEGF mRNA was associated with hypoxic zones surrounding areas of necrosis in the tumors, and that the drop in VEGF mRNA observed in tumors from mice treated with MDL 101,731 correlated with a loss of zones of necrosis. In contrast, treatment with cisplatin led to either an increase (PC-3) or no change (MDA-MB-231) in VEGF mRNA levels, and no loss of necrotic zones. Quantitative analysis of changes in VEGF mRNA levels was supported by immunohistochemical analysis of VEGF protein in the same tumor specimens. In vitro, MDL 101,731 was a potent inhibitor of VEGF secretion in cells exposed to hypoxia, whereas there was no effect of cisplatin on VEGF secretion by three of the four cell lines tested. These findings suggest that inhibition of VEGF expression by MDL 101,731 may distinguish this compound from other classes of cytotoxic agents, such as cisplatin.

Microautoradiographic quantitation of vascular endothelial growth factor mRNA levels in human prostate specimens containing normal and neoplastic epithelium.

Human prostate specimens commonly contain a spectrum of epithelial changes, including normal acinar and ductal structures, hyperplasia, intraepithelial neoplasia (dysplasia), and carcinoma. Since vascular endothelial growth factor (VEGF) expression is dependent on cell type and tissue microenvironment, meaningful quantitation of the levels of this mRNA in pathological specimens requires analysis at the microscopic level. Phosphorimage analysis of the binding of radiolabeled cRNA probes to tissue sections allows quantitation of mRNA levels, but the resolution is limited. Alternatively, emulsion autoradiography allows visualization of mRNA levels at cellular resolution, but quantitation is difficult. We have developed a method of quantitating steady state mRNA levels in tissue sections at the microscopic level, using autoradiography and quantitative image analysis. In this study, we describe the method and apply it to quantitation of VEGF mRNA in human prostate specimens. The VEGF mRNA level was low in nonepithelial stromal tissue (0.8 dpm/mm2), high in normal and benign hyperplastic epithelium (17-18 dpm/mm2), and significantly decreased in intraepithelial neoplasia (6.4 dpm/mm2) and in microacinar carcinoma that had invaded the stroma (3.5 dpm/mm2). Immunohistochemical staining detected VEGF protein in epithelial and stromal cells, with highest levels on the luminal surface of normal epithelium and in stromal cells, and lower levels in benign hyperplasia, intraepithelial neoplasia, and carcinoma. No correlation between VEGF expression in epithelium and nearby vessel density was observed. The results indicate a decrease in the steady state level of VEGF mRNA when prostate epithelial cells become transformed, escape the confines of glandular structure and invade the stroma, and suggest that the progression of prostatic carcinoma through the stages examined in this study is not associated with increased VEGF expression, in contrast to the elevated VEGF expression associated with progression of several other tumor types.

Quantitation of vascular endothelial growth factor mRNA levels in human breast tumors and metastatic lymph nodes.

In situ hybridization analysis provides a means to qualitatively study the heterogeneity of primary tumors and metastases based on the types of genes transcribed. In this study, we have tested some parameters for quantitative analysis of in situ hybridizations with paraffin-embedded human breast tumors and measured mRNA levels for the angiogenic protein, vascular endothelial growth factor (VEGF). VEGF mRNAs were highly tumor specific, with the highest levels near necrotic regions within the tissues (0.1 to 2.7 dpm/mm2). Normal cells within the tissue sections did not have detectable levels of VEGF mRNA. For comparison, tumor levels of c-myc (4 to 46 dpm/mm2) and glyceraldehyde-3-phosphate dehydrogenase mRNAs (48 to 214 dpm/mm2) were measured. The mRNAs for both of these genes were more broadly expressed across the tissue sections. The hybridization pattern for VEGF mRNAs was consistent with hypoxia-induced VEGF mRNA steady-state levels and supports the hypothesis that oxidative stress regulates VEGF expression in breast tumors.

A ribonucleotide reductase inhibitor, MDL 101,731, induces apoptosis and elevates TRPM-2 mRNA levels in human prostate tumor xenografts.

MDL 101,731, (E)2'-fluoromethylene-2'-deoxycytidine, is an irreversible inhibitor of ribonucleotide diphosphate reductase and causes regression of human tumors in nude mouse models. Messenger RNA levels for testosterone-repressed prostatic message-2 (TRPM-2), a transcript that increases in human tumor xenografts undergoing programmed cell death, were analyzed by in situ hybridization. Xenografts derived from a human prostate tumor cell line (PC-3) regressed following treatment with MDL 101,731 and the relative levels of TRPM-2 mRNA increased up to threefold in drug-treated animals. Apoptosis in the tumor xenografts was further indicated by in situ labeling of DNA strand breaks by incorporation of biotinylated-dUTP with terminal deoxynucleotidyl transferase. In vitro, PC-3 cells incubated with MDL 101,731 showed evidence of apoptosis based on flow cytometry and DNA laddering. These data support the hypothesis that MDL 101,731 stimulates programmed cell death in regressing PC-3 xenografts.