Tsga10 expression correlates with sperm profiles in the adult formalin-exposed mice.

Testis-specific gene antigen10 (Tsga10), as a cytoskeletal protein in the sperm tail, impacts the sperm motility. This study investigates the correlation between sperm profile alterations and Tsga10 gene expression in adult mice exposed to formaldehyde (FA) and then treated with antioxidant effect of manganese (Mn2+ ). In this regard, we examined 35 NMRI adult male mice (6-8 weeks age) in 4 groups of control, sham, FA-exposed and FA+Mn2+ . The mice in FA+Mn2+ group were exposed to FA (10 mg kg-1 twice a day) for 2 weeks and treated with daily Mn2+ administration (5 mg kg-1 ) in the second week prior to sacrificing the mice for testis dissection. The right testis was dissected in each group and subjected to RNA extraction and cDNA syntheses for gene expression analysis by real-time PCR. The findings revealed that FA decreased sperm parameters and Tsga10 expression (52.6 ± 24.37%). However, the injected powerful manganese antioxidant improved sperm profile through overexpression of Tsga10 (121.6 ± 27.13%) under FA-induced stressful condition which proves the correlation between sperm profile and Tsga10 expression (P ≤ 0.05). This study also shows that Tsga10 expression protects sperm dysfunction in FA+Mn2+ group and resulting in better preservation of spermatozoa and improvement of male fertility.

In vitro toxicity assay of cisplatin on mouse acute lymphoblastic leukaemia and spermatogonial stem cells.

Testicular cancer is the most common cancer affecting men in reproductive age, and cisplatin is one of the major helpful chemotherapeutic agents for treatment of this cancer. In addition, exposure of testes cancer cells to cisplatin could potentially eliminate tumour cells from germ cells in patients. The aim of this study was to evaluate the effect of cisplatin on viability of mouse acute lymphoblastic leukaemia cell line (EL-4) and neonatal mouse spermatogonial cells in vitro. In this study, the isolated spermatogonial stem cells (SSC) and EL-4 were divided into six groups including control (received medium), sham (received DMSO in medium) and experimental groups which received different doses of cisplatin (0.5, 5, 10 and 15 µg ml(-1) ). Cells viability was evaluated with MTT assay. The identity of the cultured cells was confirmed by the expression of specific markers. Our finding showed that viability of both SSC and EL-4 cells was reduced with the dose of 15 µg/ml when compared to the control group (P ≤ 0.05). Also, the differences between the IC50 in doses 10 and 15 µg/ml at different time were significant (P ≤ 0.05). The number of TUNEL-positive cells was increased, and the BAX and caspase-3 expressions were upregulated in EL4 cells for group that received an effective dose of cisplatin). In conclusion, despite the dramatic effects of cisplatin on both cells, spermatogonial stem cells could form colony in culture.

Development of anti-p185HER2 immunoliposomes for cancer therapy.

The product of the HER2 protooncogene, p185HER2, represents an attractive target for cancer immunotherapies. We have prepared anti-p185HER2 immunoliposomes in which Fab' fragments of a humanized anti-p185HER2 monoclonal antibody with antiproliferative properties (rhuMAb-HER2) were conjugated to either conventional or sterically stabilized liposomes. These immunoliposomes bind specifically to p185HER2-overexpressing breast cancer cells (SK-BR-3 and BT-474). High-affinity binding of anti-p185HER2 immunoliposomes is comparable to that of free rhuMAbHER2-Fab' or the intact antibody. Empty immunoliposomes inhibit the culture growth of p185HER2-overexpressing breast cancer cells, and this antiproliferative effect is superior to that of free rhuMAbHER2-Fab', indicating that liposomal anchoring of these anti-p185HER2 Fab' fragments enhances their biological activity. Efficient internalization of anti-p185HER2 immunoliposomes, demonstrated by light and electron microscopy, occurs by receptor-mediated endocytosis via the coated pit pathway and also possibly by membrane fusion. Doxorubicin-loaded anti-p185HER2 immunoliposomes are markedly and specifically cytotoxic against p185HER2-overexpressing tumor cells in vitro. Anti-p185HER2 immunoliposomes administered in vivo in Scid mice bearing human breast tumor (BT-474) xenografts can deliver doxorubicin to tumors. These results indicate that anti-p185HER2 immunoliposomes are a promising therapeutic vehicle for the treatment of p185HER2-overexpressing human cancers.

Echo-planar perfusion-sensitive MR imaging of acute cerebral ischemia.

T2*-sensitive echo-planar magnetic resonance imaging was used with first-pass magnetic susceptibility contrast enhancement in a cat model of acute regional stroke to evaluate the relationship between cerebral hypoperfusion and ischemic brain damage. In normal brain, dose-dependent decreases in signal intensity were observed after intravenous injection of 0.15-0.50 mmol/kg dysprosium-diethylenetriaminepentaacetic acid bismethylamide or gadodiamide injection. Shortly after unilateral occlusion of the middle cerebral artery, foci of signal hyperintensity on diffusion-weighted images were observed in the ipsilateral basal ganglia. Sixty minutes after occlusion, perfusion deficits in the ipsilateral parietal and temporal cortical gray matter were observed to be spatially correlated with areas of hyperintensity on diffusion-weighted images. When reflow was attempted after 60 minutes, delayed contrast agent transit suggestive of partial ischemic tissue injury was demonstrated. Attempts to produce reflow after 2 hours did not restore normal brain perfusion and resulted in image hyperintensity and histopathologic brain damage. Six-hour occlusion was associated with pronounced perfusion deficits in the ischemic territory.

Cerebrovascular transit characteristics of DyDTPA-BMA and GdDTPA-BMA on normal and ischemic cat brain.

PURPOSE: To compare the efficacy of two nonionic T2*-shortening contrast agents, DyDTPA-BMA dysprodiamide injection and GdDTPA-BMA gadodiamide injection, as perfusion-sensitive MR imaging agents in normal and acutely ischemic brain. METHODS: The magnetic susceptibility effects of intravenous injections of 0.10-0.50 mmol/kg of each contrast agent were quantified on T2-weighted spin-echo images of cat brain before and after unilateral occlusion of the middle cerebral artery by measuring signal intensity changes in the same regions-of-interest in parietal cortex. RESULTS: In normal brain, DyDTPA-BMA produced a significantly greater loss of signal intensity than equimolar doses of GdDTPA-BMA. The magnitude of the signal intensity attenuation was dosage-dependent and proportional to the square of the magnetic moments of the two contrast agents. Restoration of baseline image signal intensity was observed within 30 min after each injection. However, injection of GdDTPA-BMA also produced a delayed, persistent hyperintensity on T2-weighted images, presumably due to its underlying T1-shortening effect. Following unilateral occlusion of the middle cerebral artery, unenhanced T2-weighted images failed to show evidence of cerebral injury for 1.5-3 hours. Administration of 0.10-1.0 mmol/kg DyDTPA-BMA shortened the time for detection of perfusion deficits (residual hyperintensity) in 22 of 36 (61%) treated cats, often to within 30 min after arterial occlusion. DyDTPA-BMA enhancement also improved lesion conspicuity in 26 of 36 (72%) cases, and disclosed very small infarcts that were not visible on T2-weighted precontrast images. Perfusion deficits in areas of partial ischemia were seen more clearly on DyDTPA-BMA-enhanced images than after equimolar injections of GdDTPA-BMA. CONCLUSIONS: Magnetic susceptibility contrast-enhanced MR imaging enables detection of perfusion deficits associated with acute cerebral ischemia well in advance of conventional T2-weighted spin-echo MR imaging without contrast. DyDTPA-BMA appears to delineate regions of ischemic damage better than GdDTPA-BMA.

In vivo diffusion-perfusion magnetic resonance imaging of acute cerebral ischemia.

We compared the anatomic extent and severity of ischemic brain injury shown on diffusion-weighted magnetic resonance (MR) images, with cerebral tissue perfusion deficits demonstrated by a nonionic intravascular T2*-shortening magnetic susceptibility contrast agent used in conjunction with standard T2-weighted spin-echo and gradient-echo echo-planar images. Diffusion-weighted images displayed increased signal intensity in the vascular territory of the middle cerebral artery 25-40 min after permanent occlusion, whereas T2-weighted images without contrast were negative or equivocal for at least 2-3 h after stroke was induced. Contrast-enhanced T2-weighted and echo-planar images revealed perfusion deficits that were spatially closely related to the anatomic regions of ischemic tissue injury. These data indicate that diffusion-weighted MR images are very sensitive to early onset pathophysiologic changes induced by acute cerebral ischemia. Combined sequential diffusion-perfusion imaging enables noninvasive in vivo examination of the relationship between hypoperfusion and evolving ischemic brain injury.

Diffusion/perfusion MR imaging of acute cerebral ischemia.

In vivo echo-planar MR imaging was used to measure apparent diffusion coefficients (ADC) of cerebral tissues in a comprehensive noninvasive evaluation of early ischemic brain damage induced by occlusion of the middle cerebral artery (MCA) in a cat model of acute regional stroke. Within 10 min after arterial occlusion, ADC was significantly lower in tissues within the vascular territory of the occluded MCA than in normally perfused tissues in the contralateral hemisphere. Sequential echo-planar imaging was then used in conjunction with bolus injections of the magnetic susceptibility contrast agent, dysprosium DTPA-BMA, to characterize the underlying cerebrovascular perfusion deficits. Normally perfused regions of brain were identified by a dose-dependent 35-70% loss of signal intensity within 6-8 s of contrast administration, whereas ischemic regions appeared relatively hyperintense. These data indicate that sequential diffusion/perfusion imaging may be useful in differentiating permanently damaged from reversibly ischemic brain tissue.

Anisotropy in diffusion-weighted MRI.

Diffusional anisotropy of water protons, induced by nonrandom, directional barriers which hinder or retard water motion, is measurable by MRI. Faster water diffusion was observed when the diffusion-sensitizing gradient direction paralleled the long axes of white matter tracts, indicative of fewer barriers to water motion. Diffusion perpendicular to this axis was as much as four times slower. Anisotropy was seen pre- and postmortem in all axial, sagittal, and coronal planes, with and without cardiac gating. Ordering has also been observed in feline optic nerve and in human peripheral nerves. Utilization of this technique can greatly improve understanding and assessment of demyelinating disorders, of white matter infarcts and neoplasms, and of neonatal brain and spinal cord development.

Ischemic brain damage: reduction by sodium-calcium ion channel modulator RS-87476.

A novel sodium-calcium ion channel modulator, RS-87476, reduced cerebral infarct size in cats subjected to permanent unilateral occlusion of the middle cerebral artery. Cerebral injury was assessed in vivo with a combination of magnetic resonance (MR) imaging and spectroscopy for 5-12 hours after occlusion and was compared with the area of histochemically ischemic brain tissue. Compared with infarcts in placebo-treated animals, infarcts in cats given RS-87476 were reduced by an average of 70% at the lowest dose, 75% at the intermediate dose, and 88% at the highest dose. Tissue edema, observed as areas of signal hyperintensity on diffusion- and T2-weighted spin-echo images, was confined to small regions of the parietal cortex and basal ganglia in drug-treated animals. Mean plasma levels of RS-87476 at the lowest dose were 13 ng/mL initially, falling to maintenance levels of 3-5 ng/mL; at the intermediate and highest doses, plasma levels of drug were approximately five- and 20-fold greater. The drug was only slightly hypotensive. At least part of the potent cerebroprotective effects of RS-87476 result from its ability to stabilize metabolic energy reserves, reduce lactate formation in ischemic tissues, and attenuate intracerebral edema.

Ultrafast magnetic resonance imaging: diffusion and perfusion.

Echo-planar magnetic resonance imaging (MRI) can be used to measure apparent diffusion coefficients noninvasively in vivo, with scan times of 150 milliseconds or less, and to assess early ischemic effects in the feline experimental model, which has an occluded middle cerebral artery (MCA). The apparent diffusion coefficient in ischemic regions, which are identified later from vital staining, is significantly decreased from normal values within 1 hour after the MCA becomes occluded. A series of 10 echo-planar images that are progressively diffusion-weighted can be collected in 1 minute (effective TR of 6 seconds). Semilogarithmic plots of image intensity versus gradient strength factors (b values) were linear. Collecting sequential gradient echo-planar images during the passage of a bolus of contrast medium is also useful when assessing perfusion or vascular integrity before and during ischemic episodes. After intravenous injection of dysprosium-diethylene triamine penta-acetic acid-bis(methylamide), typical signal losses of 40% to 80% were observed and were dose-dependent. Areas of possible ischemia identified from diffusion-weighted images did not lose signal intensity with the use of contrast medium and were seen as regions of relative hyperintensity, clearly discernible from normally perfused tissues.

Gossypol effects on endothelial cells and tumor blood flow.

Isomers (-, +) of the antitumor agent gossypol (G) were studied for their ability to reduce tumor ATP and blood flow in rats bearing subcutaneously implanted pancreatic tumors. A 50% reduction in tumor ATP/Pi within ih of a single injection of -G was associated with a 60% decline in tumor blood flow. To determine if these changes in tumor physiology could be due to a direct drug effect on tumor endothelium, G isomers were compared for their ability to alter protein (125I-BSA) permeability and metabolic (32P) labelling of cultured endothelial cells. Treatments for ih produced no endothelial cell leakage, but 24h exposures to either -G (5 microM) or +G (50 microM) produced complete permeability of the monolayers to 125I-BSA. In contrast, 0.5-I.Oh exposures to -G (4 microM) produced 2 to 3-fold increases in phosphorylated 27 kDa heat-shock protein, hsp-27. Hsp-27 phosphoprotein isoforms were differentially labelled following -G and +G exposures with the phosphorylation profile of -G appearing most similar to that of oxyradical producing agents known to induce hsp-27 and injure endothelial cells. We postulate that the tumor ischemic effects of -G are mediated by endothelial response to oxyradical production in a mechanism similar to that of tissue ischemia-reperfusion injury.

Diffusion-weighted MR imaging of anisotropic water diffusion in cat central nervous system.

The diffusion behavior of intracranial water in the cat brain and spine was examined with the use of diffusion-weighted magnetic resonance (MR) imaging, in which the direction of the diffusion-sensitizing gradient was varied between the x, y, and z axes of the magnet. At very high diffusion-sensitizing gradient strengths, no clear evidence of anisotropic water diffusion was found in either cortical or subcortical (basal ganglia) gray matter. Signal intensities clearly dependent on orientation were observed in the cortical and deep white matter of the brain and in the white matter of the spinal cord. Greater signal attenuation (faster diffusion) was observed when the relative orientation of white matter tracts to the diffusion-sensitizing gradient was parallel as compared to that obtained with a perpendicular alignment. These effects were seen on both premortem and immediate postmortem images obtained in all axial, sagittal, and coronal views. Potential applications of this MR imaging technique included the stereospecific evaluation of white matter in the brain and spinal cord and in the characterization of demyelinating and dysmyelinating diseases.

Diffusion-weighted MR imaging of acute stroke: correlation with T2-weighted and magnetic susceptibility-enhanced MR imaging in cats.

We evaluated the temporal and anatomic relationships between changes in diffusion-weighted MR image signal intensity, induced by unilateral occlusion of the middle cerebral artery in cats, and tissue perfusion deficits observed in the same animals on T2-weighted MR images after administration of a nonionic intravascular T2 shortening agent. Diffusion-weighted images obtained with strong diffusion-sensitizing gradient strengths (5.6 gauss/cm, corresponding to gradient attenuation factor, b, values of 1413 sec/mm2) displayed increased signal intensity in the ischemic middle cerebral artery territory less than 1 hr after occlusion, whereas T2-weighted images without contrast usually failed to detect injury for 2-3 hr after stroke. After contrast administration (0.5-1.0 mmol/kg by Dy-DTPA-BMA, IV), however, T2-weighted images revealed perfusion deficits (relative hyperintensity) within 1 hr after middle cerebral artery occlusion that corresponded closely to the anatomic regions of ischemic injury shown on diffusion-weighted MR images. Close correlations were also found between early increases in diffusion-weighted MR image signal intensity and disrupted phosphorus-31 and proton metabolite levels evaluated with surface coil MR spectroscopy, as well as with postmortem histopathology. These data indicate that diffusion-weighted MR images more accurately reflect early-onset pathophysiologic changes induced by acute cerebral ischemia than do T2-weighted spin-echo images.

Early detection of ischemic injury: comparison of spectroscopy, diffusion-, T2-, and magnetic susceptibility-weighted MRI in cats.

Within one hour following MCA-occlusion in cats, heavily diffusion-weighted spin-echo MR images exhibited a well-defined hyperintensity in the gray matter and basal ganglia of the occluded side over normal side. This hyperintensity correlated with lactate and inorganic phosphate increases in peak areas from MR surface coil spectroscopy. T2-weighted MRI showed no significant abnormality in signal intensity from the occluded hemisphere within several hours post-occlusion. Using a paramagnetic MR contrast agent, dysprosium-DTPA-BMA together with heavily T2-weighted spin-echo or with T2*-weighted echo-planar (EPI) MR imaging, perfusion deficits resulting from MCA-occlusion were detected as a relative hyperintensity of ischaemic tissues compared to normally-perfused cerebral tissues in the contralateral hemisphere. Evidence of these deficits was observed within minutes of occlusion, and spatially correlated well with the hyperintensity seen on the diffusion-weighted images. Diffusion- and susceptibility-weighted MRI was superior to conventional T2-weighted MRI in the detection of early ischaemic events. In contrast to surface coil spectroscopy, both techniques mapped regions of jeopardy throughout the brain, which later showed T2-weighted hyperintensity and lack of vital (TTC) staining.

MR evaluation of calcium entry blockers with putative cerebroprotective effects in acute cerebral ischaemia.

MR imaging and spectroscopy were used to investigate whether two calcium channel entry-blockers, nicardipine and RS-87476 (Syntex), would reduce ischaemic brain damage in barbiturate-anaesthetized cats subjected to permanent unilateral occlusion of the middle cerebral artery (MCA). The evolution of cerebral injury was assessed in vivo in a total of 38 cats using a combination of diffusion-weighted and T2-weighted spin-echo proton MR imaging and phosphorus 31 (P-31) and proton (H-1) MR spectroscopy for up to 12 h following arterial occlusion. Immediately thereafter, the volume of histochemically ischaemic brain tissue was determined planimetrically. In untreated control animals, diffusion-weighted MR images obtained with strong gradient strengths (5.5 gauss/cm) displayed increased signal intensity (oedema) in the ischaemic MCA territory less than 45 min after stroke. These changes were closely correlated with the appearance of abnormal P-31 and H-1 metabolite levels evaluated with surface coil MR spectroscopy. Cats injected with i.v. nicardipine (10 micrograms/kg bolus, 8 micrograms/kg/h maintenance) or RS-87476 (2-50 micrograms/kg bolus, 0.7-17.5 micrograms/kg/h maintenance) showed a significant reduction in ischaemic injury in the ipsilateral cerebral cortex, internal capsule and basal ganglia. The results of this study suggest that these calcium entry blockers protect against brain damage induced by acute stroke by stabilizing cellular metabolic processes, reducing lactate formation in ischaemic tissues, and attenuating cytotoxic and vasogenic oedema.