Validation of image analysis for enzyme histochemical and immunocytochemical staining.

Immunocytochemical and enzyme histochemical analyses of cells and tissues are used to detect changes in the extent of injury and the expression of various molecules. Image analysis quantitation offers an easier, more efficient technique to evaluate these changes. We studied the application of image analysis for evaluating enzyme histochemistry and immunocytochemistry of cells and tissues as a way to assess stroke. Using brain sections, we compared investigator and computer-generated image analysis of 2,3,5-triphenyltetrazolium chloride stained cerebral infarcts in rats subjected to 2 h middle cerebral artery occlusion and 22 h re-perfusion. Both methods documented the infarct volumes with a comparison of means of less then 5%. This suggests no difference between computer- and hand-calculated values. Computer-generated analysis was easier and faster to use. Using endothelial cell monolayers, immunocytochemical staining of a time course of heat shock protein expression was compared to a grading system using fast red chromagen counterstained with hematoxylin. Results demonstrated greater ease and efficiency with computer-generated image analysis compared to other subjective systems of analysis. Image analysis is more useful for detecting small differences in staining, especially when using 3,3-diaminobenzidine as a chromagen. Investigator bias is also reduced using this system. Our comparisons validate the use of this versatile technology to assess more easily both cell and tissues in stroke research.

Temporal changes in sensitivity of rats to cerebral ischemic insult.

OBJECT: Experimental rat models are often used to study cerebral ischemia, yet rats are nocturnal animals that have activity cycles that are the opposite of those of humans. In the following study the authors examined the circadian rhythm of sensitivity to an ischemic insult in rats by using an intraluminal thread technique to produce reversible middle cerebral artery occlusion. METHODS: Ischemia (2 hours of blockage followed by 22 hours of reperfusion) was induced in rats according to the 24-hour clock at either 100, 400, 700, 1,000, 1,300, 1,600, 1,900, or 2,200 hours (11-14 rats per time period). The rat brains were removed, coronally sectioned, stained with 2,3,5-triphenyltetrazolium chloride and analyzed using commercially available software. Analysis of variance and cosinor-rhythmometry statistical tests were used for analysis of data. The time of day when the ischemic infarct was induced had a significant (p = 0.011) influence on the volume of the lesion. The volume of total brain infarct produced at 400 hours (7.65 +/- 1.31%) was more than three times greater than the volume produced at 1600 hours (2.1 +/- 0.34%). Cosinor-rhythm analysis indicated a peak occurrence of infarct volume at 6:02 (95% confidence interval 5:49-6:16). The size of the infarct correlated with core body temperature rhythms, which varied by 1.3 +/- 0.62 degrees C (mean +/- standard deviation). CONCLUSIONS: Circadian rhythms, as well as the reversed natural body rhythms of the rat compared with humans, should be considered when extrapolating data to human or other animal studies. Temporal rhythms may also provide information concerning the cascading disease processes associated with cerebral ischemia.

Endovascular management of aneurysm and carotid-cavernous fistulae from gunshot wounds to the skull base and oropharynx.

The clinician must be aware of the potential for vascular injury that can result from gunshot wounds to the skull base and oropharynx. These lesions can be life-threatening or can result in irreversible neurologic defects. The goal is early diagnosis and efficient appropriate treatment. Endovascular therapy has been proven to be of great benefit for the treatment of traumatic aneurysms and carotid-cavernous fistulae. Utilizing either a reconstructive or a deconstructive approach, the traumatic lesions can be treated without the morbidity inherent to surgery of the skull base or cavernous sinus. This article discusses the authors' experience with endovascular treatment, explaining in detail the reconstructive and deconstructive approaches and providing clinical examples of the treatment of pseudoaneurysms and carotid-cavernous fistulae.

Monocyte adherence to the subendothelial basement membrane increases interleukin-8 gene expression and antigen release.

The emigration of peripheral blood monocytes into the interstitium allows for contact with a variety of surfaces which may provide signals important for monocyte function in both normal and inflammatory states. In the present study, we examined the effect of adherence to an endothelial cell-derived basement membrane and to collagen I, the major collagen of the interstitium, on monocyte release and gene expression of the potent chemotactic cytokine Interleukin-8 (IL-8). We further evaluated neutrophil chemotactic activity of the conditioned media containing antigenic IL-8 from monocytes adherent to these same surfaces. Elutriation-purified monocytes were adhered for 1 hour to plastic tissue culture wells either uncoated (PL) or coated with bovine serum albumin (BSA), collagen type I (C-I), or endothelial cell-derived basement membrane (BM). Following removal of nonadherent cells, monocytes were further incubated in a serum-free media for 18 hours in the presence or absence of lipopolysaccharide (IPS). Following 18 hrs of incubation there were significantly less monocytes remaining adherent to BM when compared to other surfaces tested. In the absence of LPS, adherent monocytes released significant amounts of IL-8 that was not surface specific. In the presence of LPS, monocytes adherent to BM released significantly more IL-8, when corrected for adherent cell number, than monocytes adherent to PL, BSA, or C-I. Conditioned media from adherent monocytes expressed IL-8 dependent neutrophil chemotactic activity that was not influenced by the surfaces tested. Northern blot analysis indicated greater induction for IL-8 mRNA by monocytes adhered to BM after 18 hrs in the presence of LPS. These results suggest that monocyte adherence to the subendothelial basement membrane provides a priming signal for the induction and secretion of the chemotactic cytokine IL-8 in response to inflammatory stimuli.

Interaction of fibronectin (FN) cell binding fragments and interleukin-8 (IL-8) in regulating neutrophil chemotaxis.

This study investigated the possible interaction of FN fragments in regulating IL-8-mediated neutrophil chemotaxis in vitro using Neuroprobe microchambers. Human neutrophil suspensions were incubated with purified FN fragments or an RGD-containing peptide and allowed to migrate in response to chemotactically active concentrations of human recombinant IL-8. The 120-kD fragment of FN containing the RGD sequence or an RGD peptide (GRGDSP) inhibited IL-8-mediated neutrophil chemotaxis; however, these RGD peptides did not inhibit neutrophil chemotaxis in response to other chemotactic agents. Furthermore, FN fragments not containing the RGD sequence had no effect on IL-8-mediated chemotaxis. These data suggest that directed migration of neutrophils in response to IL-8 is inhibited in the presence of cell-binding fragments of FN and may represent a local mechanism for terminating neutrophil migration at areas of tissue injury.

Priming of human monocyte superoxide production and arachidonic acid metabolism by adherence to collagen- and basement membrane-coated surfaces.

Monocytes (m phi s) come into intimate contact with basement membranes and extracellular matrix proteins as they extravasate from the blood to the interstitium or to sites of tissue injury. We examined the in vitro effects of m phi adherence to an endothelial cell-derived basement membrane or to purified extracellular matrix proteins on phorbol myristate acetate (PMA)-stimulated superoxide production and prostanoid secretion. Elutriation-purified human peripheral blood m phi s were adhered to tissue culture wells that were precoated with the following purified proteins: bovine serum albumin (BSA), collagen type I (COL I), collagen type IV (COL IV), fibronectin (FN), or laminin (LM). To model the provisional matrix at sites of tissue injury, m phi s were also adhered to wells coated with either denatured collagen type I or gelatin (GEL) or coated with basement membrane (BM) derived from endothelial cell monolayers. The m phi s were adhered to the protein-coated surfaces for 1 h at 37 degrees C in serum-free medium and washed to remove nonadherent cells, and the number of adherent m phi s was measured. Monolayers of m phi s were also incubated for an additional 18 h, at which time both adherence and cell spreading were measured. PMA-stimulated superoxide production by adherent m phi s was determined after 1 and 18 h of adherence to the protein-coated surfaces. PMA-stimulated release of two prostanoids, prostaglandin E2 (PGE2) and thromboxane B2 (TxB2) was measured after 18 h of m phi adherence to the surfaces. Following 18 h of adherence, PMA-stimulated superoxide anion secretion and secretion of PGE2 and TxB2 were augmented of primed by m phi s adherent to COL I, GEL, or BM. In contrast, no such priming effects were observed by m phi s adherent to COL IV, FN, or LM. The results suggest that adherence to basement membranes, collagen type I-containing surfaces in the interstitium, or denatured collagen at sites of tissue injury primes m phi respiratory burst and arachidonate metabolism to inflammatory agonists. Induction of priming events by substrate-specific adherence may be an important factor regulating host defense functions of m phi s in the extracellular matrix.

Leukocyte involvement in cerebral infarct generation after ischemia and reperfusion.

White blood cell involvement in the generation of cerebral infarcts was evaluated following ischemia and reperfusion injury in the rat. Control and leukopenic rats (induced by vinblastine, WBC counts < 1500/mm3) were compared in a global forebrain ischemic model after 1 h of ischemia and 1 h 15 min of reperfusion. Cerebral infarcts were defined on coronal brain sections using Triphenyl tetrazolium chloride (TTC) staining. Electroencephalographic activity (EEG) and somatosensory evoked potentials (SSEP) were also compared. Results indicate that the area infarcted in leukopenic rats was significantly less than infarcts generated in corresponding controls (21 +/- 16% vs. 70 +/- 16%). In addition, EEG was preserved in all leukopenic animals when compared to controls, both during ischemia and after reperfusion. The cortical peak component of the SSEP was also better preserved in the leukopenic animals both during ischemia and at 60 min of reperfusion. These results indicate white blood cell participation in the generation of cerebral damage in a model of global forebrain ischemia and reperfusion as indicated by TTC staining of cerebral infarcts.

Leukocyte involvement in cerebral ischemia and reperfusion injury.

Leukocytes have been postulated to contribute to cerebral ischemia and reperfusion injury. The present study implies that leukocytes have a deleterious effect in the brain following ischemia. We compared the alteration of cortical electrical activity following transient, incomplete cerebral ischemia in control and leukopenic rats by monitoring somatosensory evoked potentials and electroencephalographic activity. There was complete cessation of electroencephalographic activity, and the cortical peak of the evoked potential was abolished during ischemia in the control animals. However, when the animals were rendered leukopenic, there was maintenance of electroencephalographic activity with reduced amplitude and preservation of the cortical peak of the evoked response during the ischemic period. This indicates that when the animals are made leukopenic, even under ischemic conditions, the neurophysiologic functioning is still maintained to a certain extent.

The character of the atrial natriuretic response: pressure and volume effects.

Synthetic atrial natriuretic factor (ANF) was compared to the well-characterized diuretics hydrochlorothiazide and furosemide in rats. While ANF was markedly more potent than either agent, its natriuretic potency more closely resembled hydrochlorothiazide. Probenecid, a drug that competes with diuretics such as furosemide for secretion into the renal tubular lumen and thereby decreases diuretic effectiveness, did not interfere with the natriuretic actions of ANF. In rats, continuous infusion of ANF resulted in a bell-shaped dose-response relationship. Above a dose of approximately 100 pmol/kg per min, the response waned and at the highest doses studies, 1-2 nmol/kg per min, urinary sodium excretion fell below control levels. A similar bell-shaped dose-response curve was observed in anaesthetized volume-expanded monkeys. During euvolaemia, conscious monkeys showed a marked fall in blood pressure without concomitant natriuresis. The blood pressure was sustained for the 3-h duration of the ANF infusion. One percent body weight volume expansion blunted the fall in blood pressure in these monkeys and the restored natriuretic response did not wane during 3-h infusion. When the fall in blood pressure was prevented by angiotensin II infusion in conscious euvolaemic monkeys, the natriuretic response to ANF was expressed.

Age-related changes in regional blood flow in the rat.

The purpose of this investigation was to characterize the changes in regional blood flow and central hemodynamic measures that occur in the rat as a result of the aging process. The isotope-labeled microsphere technique was used to measure cardiac output and regional blood flows in conscious and anesthetized adult (12 mo) and senescent (24 mo) Fischer 344 virgin female rats. No significant changes were observed in central hemodynamic measurements or regional blood flows in conscious rats with the exception of a 25% reduction in splenic blood flow. Pentobarbital anesthesia significantly reduced cardiac index and heart rate but elevated total peripheral resistance and mean arterial blood pressure. There was a decrease in blood flow to skeletal muscle, spleen, duodenum, stomach, and brain tissue samples and increased hepatic arterial blood flow in both age groups. The use of anesthesia caused a greater reduction in the cardiac index and brain blood flow in the senescent anesthetized rats than in the adult rats. Heart and kidney blood flows were decreased by anesthesia in the senescent rats but not in the adult rats. Skeletal muscle blood flow, however, was significantly greater in the senescent anesthetized rats than in the younger anesthetized animals. Although body weight and organ weights of the liver, spleen, kidneys, stomach, heart, and brain were significantly greater for the senescent rats, no differences could be demonstrated in tibial length or lean body mass.

Specific membrane receptors for atrial natriuretic factor in renal and vascular tissues.

Membranes from rabbit aorta and from rabbit and rat kidney cortex possess high-affinity (Kd = 10(-10) M) specific binding sites for atrial natriuretic factor (ANF). Similar high-affinity sites are present in an established cell line from pig kidney, LLC-PK1. Results of fractionation studies indicate that the receptors are localized in the plasma membrane of these tissues. The binding is time-dependent and saturable. An excellent quantitative correlation was found between the affinity of synthetic ANF and analogs of intermediate activity to aorta membranes and the half-maximal concentration needed for relaxation of rabbit aorta rings contracted by addition of serotonin. Furthermore, the binding affinity of the receptor in kidney membranes is consistent with the concentration required for in vivo natriuresis in the rat. Biologically inactive synthetic ANF fragments and other peptide hormones such as angiotensin II and vasopressin do not significantly inhibit binding. These data suggest that the receptors for ANF in vascular and renal tissues are responsible for mediating the physiological actions of this peptide in these target tissues.