Mouse model of microembolic stroke and reperfusion.

BACKGROUND AND PURPOSE: To test the role of fibrinolysis in stroke, we used a mouse model in which preformed 2.5- to 3-microm-diameter fibrin microemboli are injected into the cerebral circulation. The microemboli lodge in the downstream precapillary vasculature and are susceptible to fibrinolysis. METHODS: We injected various doses of microemboli into the internal carotid artery in mice and characterized their distribution, effects on cerebral blood flow, neurological deficit, infarct area, and spontaneous dissolution. By comparing wild-type and tissue plasminogen activator (tPA) knockout (tPA-/-) mice, we analyzed the role of endogenous tPA in acute thrombotic stroke. RESULTS: Microemboli cause dose-dependent brain injury. Although moderate doses of microemboli are followed by spontaneous reperfusion, they result in reproducible injury. Gene knockout of tPA markedly delays dissolution of cerebral emboli and restoration of blood flow and aggravates ischemic thrombotic infarction in the brain. CONCLUSIONS: We describe a microembolic model of stroke, in which degree of injury can be controlled by the dose of microemboli injected. Unlike vessel occlusion models, this model can be modulated to allow spontaneous fibrinolysis. Application to tPA-/- mice supports a key role of endogenous tPA in restoring cerebral blood flow and limiting infarct size after thrombosis.

In vitro properties and organ uptake of rat band 3 cross-linked erythrocytes.

Chemical conditions for cross-linking rat erythrocytes with bis(sulfosuccinimidyl)suberate (BS3) and 3,3' dithiobis(sulfosuccinimidyl propionate) (DTSSP) have been studied. These two cross-linking reagents seem to react with band 3 proteins in rat erythrocyte membrane. Two different conditions have been assayed using different cell/cross-linker concentration ratios. Similar cell volumes were observed in cross-linked rat erythrocytes compared to rat control erythrocytes. Cell yields after cross-linking account for about 65% when a high ratio of cell-to-cross-linker was used. Slightly lower cell yields (about 62%) were obtained when this ratio was reduced. Estimation of band 3 cross-linking by gel electrophoresis revealed a level of cross-linking of around 45% and 50% at the different conditions used. In vivo behavior of these modified rat erythrocytes revealed that they do not circulate, showing a predominant localization in the liver. This effect is evident from the concentration (5 mM BS3 or DTSSP) used. Based on these results, BS3 and DTSSP can be considered as useful tools to cross-link rat erythrocyte band 3 and to target rat erythrocytes to the liver.

Local and systemic effects of water-soluble contrast media and barium in rats with chronic small bowel obstruction.

RATIONALE AND OBJECTIVES: The local effects on the small intestine and systemic changes produced by different contrast media in small bowel obstruction, with time courses of 4 days, were evaluated. MATERIALS AND METHODS: Four groups, each with 10 normal rats and another four groups (also each with 10 rats) that had ligation of the terminal ileum (obstructed rats) for 4 days were given 3 mL of barium, meglumine sodium diatrizoate, iohexol, or saline (control animals). Radiographs were taken immediately, 1 and 4 hours after administration of contrast media. Immediately before sacrifice, blood samples were taken to determine the hematocrit (Hct), hemoglobin (Hb), white blood cell count (WBC), red blood cell count (RBC), and serum sodium, and potassium and chloride concentrations. Specimens of small bowel were taken for histologic and morphometric analysis. RESULTS: In obstructed rats, the image quality with iohexol improved on final radiographs despite being diluted in the great intestinal contents. There was an improvement in the serum electrolyte concentrations in the obstructed animals that were given any one of the contrast media, the best improvement being in the iohexol groups. A shortening of the length of epithelial cells when any one of the contrast media was administered was observed, as was an increase in the lymphatic space area in the diatrizoate group in normal rats. In the bowel proximal to the obstruction, the lymphatic space area was increased in the diatrizoate group and the size of the epithelial cells was higher in the diatrizoate and iohexol groups compared to the barium and saline groups. CONCLUSION: Our results suggest that iohexol offers good radiologic efficacy and excellent systemic and local tolerance in small bowel obstruction.

Plasma levels and urinary excretion of iodine after oral administration of iohexol in dogs and cats.

RATIONALE AND OBJECTIVES: Plasma and urinary iodine concentrations were assessed after oral administration of iohexol in five dogs and five cats. MATERIALS AND METHODS: Ventrodorsal and right lateral radiographs were taken; a 3-mL blood sample was collected 30 minutes and 1, 2, 4, and 6 hours after dose in each animal; and the bladder was emptied at 2 and 6 hours in dogs and at 6 hours in cats after oral administration of 700 mg I/kg iohexol (300 mg I/mL). Iodine concentration was measured using a sequential inductively coupled plasma atomic emission spectrometer system. RESULTS: Peak concentrations of plasma iodine were detected in samples taken from both dogs (0.33 mg I/mL) and cats (0.28 mg I/mL) 30 minutes after iohexol administration, with a gradual decline at 1, 2, 4, and 6 hours. Total urinary excretion during the 6 hours after the administration does of iohexol was higher (4.3%) in cats than in dogs (2.0%). In each species, there was a relationship between gastrointestinal motility observed radiographically and the subsequent excretion of iodine in the urine. CONCLUSIONS: Results indicate that approximately 2% of the oral dose is absorbed and excreted in the urine of dogs within 6 hours, and approximately 4% of the dose is absorbed and excreted in cats.

In vivo survival and organ uptake of loaded carrier rat erythrocytes.

Rat RBCs loaded with 125I-CA by hypotonic dialysis and isotonic resealing were evaluated as a carrier system. Loaded RBCs stored at 4 degrees C remained unlysed (90% survival) allowing release of encapsulated 125I-CA for up to 4 days. Thereafter, cellular lysis increased significantly. IP-injected loaded RBCs reached the maximum level (50%) in circulation at 24 h post-injection. Circulating loaded RBCs showed a half-life of 8-10 days, which was advantageous for carrier function. In contrast to IP-injected free CA, which remained in circulation for only a short time, encapsulated CA showed significant levels in circulation up to 10 days post-injection. The profile of organ uptake with time is essentially not altered for loaded with respect to native cells, being higher the removal of loaded cells and mainly localized in spleen. Nevertheless, liver is the organ with highest elimination capacity for both native and loaded cells, showing its maximum at 24 h post-injection. Concomitantly, the concentration of 125I-CA in all organs studied was highest at this time. These data demonstrate that rat loaded RBCs can potentially be used as a carrier system for long-term dissemination of drug into the organism, with specially increased delivery to the spleen. They also support the use of the rat as an experimental model for biochemical and pharmacological studies in these therapeutic systems.

Method of recording cerebrospinal fluid pressure at three anatomic places in conscious and unrestrained adult wistar rats.

There does not appear to be a consensus on the normal values of cerebrospinal fluid pressure (Pcsf) in rats because of the different conditions in which each experiment has been done. Anaesthesia, restraint or recordings from different anatomical places and the use of simultaneous infusions could produce a great variability in the obtained results and because of that the average Pcsf in rats has been reported to be between 2 and about 18.5 cm H2O. The aim of this study is to provide new information about the normal values of Pcsf in adult, conscious rats by carrying out long-term measurements, for 3 consecutive days, from different anatomical places. Thirty male Wistar rats divided in three experimental groups of ten animals each were used. A catheter of silicone was implanted into the lateral ventricles in group I, the cisterna magna in group II, and the lumbar subarachnoid space in group III to record the Pcsf values. This catheter was attached to a swivel system to allow unrestrained and free behaviour in the rats. Mean values of normal Pcsf values were 13.03 +/- 0.73 cm H2O from the lateral ventricles, 15.9 +/- 0.73 cm H2O from the cisterna magna, and 16.73 +/- 1.14 cm H2O from the lumbar subarachnoid space.

Influence of aerobic oxidation of mouse erythrocytes on their recognition by macrophages.

Membrane protein modification can change cell surface properties which can be correlated with altered macrophage-erythrocyte interactions. Mouse erythrocytes were incubated in phosphate buffer for different times to induce protein modification. Mouse erythrocyte membrane changes were analyzed by infrared analyses and gel electrophoresis. Proteolytic digestion of membrane proteins was observed. After 22 hours preliminary incubation, the number of erythrocytes adhering to a monolayer of macrophages reached a maximum, the majority of which had not been phagocytosed. Most of the erythrocytes incubated for 40 hours underwent phagocytosis after adhesion to the macrophages.

Comparison of different doses of iohexol with amidotrizoate for excretory urography in cats.

In five cats with normal renal function, doses of 200, 400, 600 and 800 mg iodine kg(-1)bodyweight of iohexol (350 mg iodine ml(-1)) were assessed in comparison to a dose of 880 mg iodine kg(-1)bodyweight of meglumine-sodium amidotrizoate (370 mg iodine ml(-1)) to determine the smallest dose which produces diagnostically adequate results for excretory urography. Urographic quality, haematologic and biochemical parameters, urinalysis and urinary osmolality, pulse and respiratory rates, blood pressure and adverse effects were determined. Iohexol presented fewer adverse reactions and influenced blood pressure less than amidotrizoate. The smallest dose of iohexol which provided urograms of similar quality to amidotrizoate was 400 mg iodine kg(-1)bodyweight. This study suggests that iohexol is safer and produces urograms of better quality than amidotrizoate.

Clinical and radiographic study of bone and joint lesions in 26 dogs with leishmaniasis.

Twenty-six dogs with parasitologically confirmed leishmaniasis and abnormalities of gait were studied to determine the most common radiological patterns of bone and joint lesions. The clinical findings included either lameness, joint pain and crepitation, soft tissue swelling and/or muscle atrophy. Bone lesions were observed radiographically in 12 of the 26 dogs; the radius and ulna were affected in seven, the tibia in six and the femur in six. Joint lesions were observed radiographically in 15 of the 26 dogs; the carpus and stifle were affected in all 15, and the tarsus in nine. There was a tendency for the bones and joints to be affected bilaterally. The radiographic patterns observed were different in the long bones and the joints. In the long bones, the most common pattern was periosteal and intramedullary proliferation, involving the diaphyses and related to the nutrient foramen; in the joints, two patterns, either non-erosive or erosive polyarthritis with soft-tissue swelling, were observed. The changes observed in the synovial fluid were associated in most cases with osteolytic lesions. However, Leishmania organisms were identified in the synovial fluid from joints without bony radiographic changes.

Comparison of the analgesic effects of meloxicam and carprofen administered preoperatively to dogs undergoing orthopaedic surgery.

Thirty-two dogs undergoing operations to repair a torn cranial cruciate ligament or a fractured long bone were randomly allocated to one of two treatment groups in a study on postoperative pain. Sixteen of the dogs were given 4 mg/kg carprofen and the other 16 were given 0.2 mg/kg meloxicam subcutaneously before the operation. The signs of pain shown by the animals were assessed for 24 hours on a visual analogue scale, a discontinuous scoring system, and a score based on five behavioural and physiological variables. The dogs' heart and respiratory rates and their mean arterial blood pressures were also measured non-invasively at each assessment. Blood samples were taken before the surgery and 24 hours after it, and the concentrations of urea and creatinine were measured in plasma. Both drugs were effective in relieving the signs of pain for up to 24 hours in all the dogs. There were no significant changes in the concentrations of urea and creatinine, and no adverse effects were reported during the postoperative period.

Unilateral renal agenesis associated with additional congenital abnormalities of the urinary tract in a Pekingese bitch.

An eight-month-old Pekingese bitch with urinary incontinence was found to have three congenital anomalies of the urinary tract: left renal agenesis, bilateral ectopic ureters with a left cranial blind-ending ureter, and urinary bladder hypoplasia. The diagnoses were made by retrograde vaginourethrography, excretory urography, ultrasonography and duplex Doppler ultrasonography. Although urological anomalies associated with renal agenesis have been frequently observed, a cranial blind-end ectopic ureter has not, to the authors' knowledge, been described in the bitch. The dog was managed medically with a restricted protein diet because of a compromised unilateral kidney with hydronephrosis and hydroureter.

Clot penetration and retention by plasminogen activators promote fibrinolysis.

Tissue-type plasminogen activator (tPA) remains the sole thrombolytic approved by the FDA for the treatment of pulmonary embolism (PE). tPA has not been replaced by third generation plasminogen activators, e.g. Reteplase (Ret) and Tenecteplase (TNK) that circulate with longer life-spans and in theory should have more extended potency in vivo. One reason for this paradox is the inability to assign units of activity to plasminogen activators based on specific biologically relevant standards, which impairs objective comparison. Here, we compare clot permeation, retention and fibrinolytic activities of tPA, TNK and Ret in vitro and clot composition over time with outcome in a mouse model of disseminated pulmonary microembolism (ME). When clots were incubated in the continuous presence of drug, tPA, TNK and Ret lysed fibrin clots identically in the absence of PA inhibitor-1 (e.g. PAI-1). Ret, which has lower fibrin affinity and greater susceptibility to inhibition by PAI-1 than tPA, was less effective in lysing plasma clots, while TNK was less effective when the fibrin content of the clots was enhanced. However, when clots were afforded only brief exposure to drug, as occurs in vivo, Ret showed more extensive clot permeation, greater retention and lysis than tPA or TNK. These results were reproduced in vivo in a mouse model of ME. These studies indicate the need for more relevant tests of plasminogen activator activity in vitro and in vivo and they show that clot permeation and retention are important potential predictors of clinical utility.

Attachment of antibody to biotinylated red blood cells: immuno-red blood cells display high affinity to immobilized antigen and normal biodistribution in rats.

Streptavidin-mediated attachment of biotinylated antibodies (b-Ab) to biotinylated red blood cells (b-RBC) is useful for preparation of immuno-red blood cells, a prospective vehicle for drug targeting. However, streptavidin (SA) induces lysis of extensively biotinylated RBC by complement due to cross-linking and inactivation of RBC complement regulators. To reduce cross-linking of RBC membrane proteins, we utilized mild biotinylation of RBC with 20 microM biotin ester (b20-RBC). SA effectively binds to rat b20-RBC (10(5) SA molecules/cell) and provides for following attachment of 5 x 10(4) molecules of b-IgG/SA per b20-RBC. By in vitro assay, b-Ab/SA/b20-RBC were stable in fresh rat serum. Serum-stable immuno-red blood cells (b-Ab/SA/b20-RBC) specifically bound to antigen-coated surfaces, but not to BSA-coated surfaces. Biodistribution of 51Cr-labelled b-Ab/SA/b20-RBC in rats was similar to that of control RBC, with no indication of lysis in vivo. These results suggest b-Ab/SA/b20-RBC may be explored as a vehicle for drug targeting.

Regulation of the complement-mediated elimination of red blood cells modified with biotin and streptavidin.

Red blood cells (RBC) modified with biotin and streptavidin (SA) present an interesting potential drug delivery system. Biotinylation and SA attachment, however, alter the biocompatibility of RBC. We have reported that polyvalent SA attachment induces lysis of biotinylated RBC (b-RBC) by homologous complement via the alternative pathway. Lysis occurs due to inactivation of the membrane regulators of complement, DAF and CD59, cross-linked by SA. However, monovalent SA attachment does not induce lysis. On the basis of these findings we hypothesized that reduction of the biotin surface density on b-RBC would allow for monovalent SA attachment to b-RBC and that such SA/b-RBC should then be stable in the circulation. In the present work we injected into rats several different radiolabeled RBC probes: rat RBC biotinylated to varying degrees (bn-RBC, where bn represents the input micromolar concentration of biotinylating agent), as well as SA/bn-RBC. Extensively biotinylated rat RBC (b700-RBC, stable in serum in vitro) were rapidly cleared from the bloodstream. We further found that extensively biotinylated human b1000-RBC bound C3b from serum in vitro without detectable lysis, and that rat b700-RBC bound to isolated macrophages in a complement-dependent fashion. Therefore, nonlytic C3b flxation and uptake of C3b-carrying b700-RBC by macrophages appears to be the mechanism leading to clearance of b700-RBC in vivo. Moderately biotinylated RBC (b70-RBC and b240-RBC) were stable in serum in vitro. SA attachment to b240-RBC led to their rapid lysis in serum in vitro, lysis in the bloodstream, and clearance by the liver and spleen. SA attachment to b70-RBC led to fast elimination of SA/b70-RBC from the bloodstream, while in vitro SA/ b70-RBC were stable in serum. Modestly biotinylated RBC (b22-RBC) demonstrated only marginally decreased 60-min survival in the bloodstream regardless of SA attachment. Our in vitro studies indicate that b23-RBC bound approximately 10(5) SA molecules per cell, and the resulting SA/b23-RBC bound 5 x 10(4) molecules of biotinylated IgG (b-IgG) per cell. About 60% of the injected dose of b-IgG/SA/b23-RBC labeled with 51Cr was detected in the rat blood cells 1 day after iv injection. To assess whether b-IgG/SA/b23-RBC circulate in the bloodstream as a stable complex, we have injected 125I-labeled b-IgG/ SA/51Cr-labeled b23-RBC in rats. Up to 60 min after injection, both radiolabels display similar level in bloodstream. Up to 3 h after injection, about 70% of 125I was detected in the blood cells. In contrast, 100% of 125I was detected in plasma after injection of nonconjugated 125I-labeled b-IgG. Thus, major portion of SA/b23-RBC-attached b-IgG circulates as a complex with RBC. About 30% of RBC-bound b-IgG undergoes detachment from the carrier b-RBC, probably in the pulmonary capillaries, because lung level of 125I was twice as high as that of 51Cr. Therefore, the surface density of biotin on the b-RBC membrane appears to play a key role in regulating complement-mediated clearance of bn-RBC and SA/bn-RBC from the bloodstream. Modest biotinylation generates b-IgG/SA/b23-RBC circulating for several hours as stable immunoerythrocytes without detectable lysis or marked elimination, and it may be possible to use these RBC in a drug delivery system.

Comparative study of air coagulation, fibrin sealant, and suture in experimental liver injury.

OBJECTIVE: To test the effects of hot air coagulation, fibrin sealant, and horizontal mattress sutures on haemostasis and regeneration in experimental hepatectomy. DESIGN: Randomised laboratory experiment. SETTING: Teaching hospital, Spain. MATERIAL: 200 rats divided into four groups (three experimental [n=60 in each] and one control [n=20]). INTERVENTIONS: Hepatic injuries were repaired by suture, coagulation, or fibrin sealant in the three experimental groups. The control group was used only to supply baseline blood samples. 10 animals in each experimental group were killed at 3, 5, 10, 25, 40, and 60 days. MAIN OUTCOME MEASURES: Time taken to achieve haemostasis, and histopathological scores of healing. RESULTS: Mattress sutures took mean (SEM) of 346 (7) seconds to control the haemorrhage and allow the liver to regain its shape and 4 rats developed abscesses (7%). Fibrin sealant achieved haemostasis immediately and the liver regained its shape in 58 (2) seconds; 2 rats (3%) developed abscesses. Hot air coagulation achieved haemostasis in 27 (1) seconds and there were no abscesses. CONCLUSION: Fibrin sealant was the best technique because it achieved immediate haemostasis and speedy regeneration. However, hot air coagulation is a useful and cheaper alternative.

Vascular immunotargeting to endothelial surface in a specific macrodomain in alveolar capillaries.

A novel 85 kD glycoprotein (gp85) is a marker of the avesicular zone, a thin part of pulmonary endothelial cells separating alveolar and vascular compartments and lacking vesicles. This report presents the first evaluation whether mAb 30B3, a monoclonal antibody to gp85, can be used for targeting of drugs to the surface of lung endothelium. 125I-mAb 30B3 accumulated in isolated perfused lungs (IPL) (22.8 +/- 1.1 versus 0.5 +/- 0.1 %ID/g for 125I-IgG) and accumulated preferentially in the lungs after intravenous or intraarterial injection (10.9 +/- 0.7 and 11.0 +/- 1.5 versus 0.9 +/- 0.2 %ID/g for 125I-IgG). 125I-mAb 30B3 uptake in IPL was rapid (T1/2 15 min), saturable (Bmax appr. 10(5) molecules/cell), specific (inhibited by nonlabeled mAb 30B3) and temperature independent (26.3 +/- 2.1 versus 22.8 +/- 1.1 %ID/g at 6 degrees C versus 37 degrees C). Biotinylated mAb 30B3 permitted subsequent accumulation of perfused avidin derivative in IPL. Because these data indicated that mAb 30B3 binds to an accessible, poorly internalizable antigen in the lung, we conjugated mAb 30B3 with a plasminogen activator, 125I-tPA. After intravenous injection in rats, lung-to-blood ratio was 8.4 +/- 0.9 for mAb 30B3/125I-tPA versus 0.4 +/- 0.1 for IgG/125I-tPA, indicating that mAb 30B3 may deliver drugs, which was supposed to exert therapeutic action in the vascular lumen (e.g., antithrombotic proteins), to the surface of pulmonary endothelium.

Cell-selective intracellular delivery of a foreign enzyme to endothelium in vivo using vascular immunotargeting.

Vascular immunotargeting, the administration of drugs conjugated with antibodies to endothelial surface antigens, has the potential for drug delivery to the endothelium. Our previous cell culture studies showed that biotinylated antibodies to PECAM-1 (a highly expressed endothelial surface antigen) coupled with streptavidin (SA, a cross-linking protein that facilitates anti-PECAM internalization and targeting) may provide a carrier for the intracellular delivery of therapeutic enzymes. This paper describes the PECAM-directed vascular immunotargeting of a reporter enzyme (beta-galactosidase, beta-Gal) in intact animals. Intravenous injection of [125I]SA-beta-Gal conjugated with either anti-PECAM or IgG led to a high 125I uptake in liver and spleen, yet beta-Gal activity in these organs rapidly declined to the background levels, suggesting rapid degradation of the conjugates. In contrast, anti-PECAM/[125I]SA-beta-Gal, but not IgG/[125I]SA-beta-Gal, accumulated in the lungs (36.0+/-1.3 vs. 3.9+/-0.6% injected dose/g) and induced a marked elevation of beta-Gal activity in the lung tissue persisting for up to 8 h after injection (10-fold elevation 4 h postinjection). Using histochemical detection, the beta-Gal activity in the lungs was detected in the endothelial cells of capillaries and large vessels. The anti-PECAM carrier also provided 125I uptake and beta-Gal activity in the renal glomeruli. Predominant intracellular localization of anti-PECAM/SA-beta-Gal was documented in the PECAM-expressing cells in culture by confocal microscopy and in the pulmonary endothelium by electron microscopy. Therefore, vascular immunotargeting is a feasible strategy for cell-selective, intracellular delivery of an active foreign enzyme to endothelial cells in vivo, and thus may be potentially useful for the treatment of acute pulmonary or vascular diseases.

Urokinase mediates fibrinolysis in the pulmonary microvasculature.

The role of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) in fibrinolysis remains unsettled. The contribution of uPA may depend on the vascular location, the physical properties of the clot, and its impact on tissue function. To study the contribution of urokinase within the pulmonary microvasculature, a model of pulmonary microembolism in the mouse was developed. Iodine 125 ((125)I)-labeled fibrin microparticles injected intravenously through the tail vein lodged preferentially in the lung, distributing homogeneously throughout the lobes. Clearance of (125)I-microemboli in wild type mice was rapid and essentially complete by 5 hours. In contrast, uPA(-/-) and tissue-type plasminogen activator tPA(-/-) mice, but not uPAR(-/-) mice, showed a marked impairment in pulmonary fibrinolysis throughout the experimental period. The phenotype in the uPA(-/-) mouse was rescued completely by infusion of single chain uPA (scuPA). The increment in clot lysis was 4-fold greater in uPA(-/-) mice infused with the same concentration of scuPA complexed with soluble recombinant uPAR. These data indicate that uPA contributes to endogenous fibrinolysis in the pulmonary vasculature to the same extent as tPA in this model system. Binding of scuPA to its receptor promotes fibrinolytic activity in vivo as well as in vitro. The physical properties of fibrin clots, including size, age, and cellular composition, as well as heterogeneity in endothelial cell function, may modify the participation of uPA in endogenous fibrinolysis. (Blood. 2000;96:1820-1826)