Microvascular response to locally injected collagenase. An experimental investigation in hamsters and rabbits.

Collagenase was injected into the ears of rabbits and the cheek-pouches of hamsters. The acute and long-term microvascular effects were studied by vital microscopy and microangiography. The enzyme was injected at three different concentrations: 120 units/ml, 600 units/ml and 3,000 units/ml. The medium (600 units/ml) and high (3,000 units/ml) concentrations induced effects on the microcirculation such as blood flow impairment and microbleedings. The magnitude of these effects was related to the concentration of the enzyme. Generally, these microvascular effects were of low magnitude as compared with other substances tested using the same experimental models.

Comparative light, scanning, and transmission electron microscopic study of chemically induced premalignant lesions in hamster's cheek pouch.

The early chemically induced epithelial dysplastic changes in the hamster's cheek pouch were studied using light, scanning, and transmission electron microscopy. Epithelial dysplasia was noticed on the light microscopic level by the sixth week of the experiment and became marked by the eighth week. At the scanning electron microscopic level, surface alterations with features previously reported in early epithelial dysplasis in human and oral mucosa were seen by the second week of the experiment and progressed over time. These early changes were also confirmed at the ultrastructural level. The usefulness of scanning and transmission microscopy in detecting early oral epithelial dysplastic changes in this animal model is discussed.

Modulation of arteriolar blood flow by inhibitors of arachidonic acid oxidation after thermal injury: possible role for a novel class of vasodilator metabolites.

To examine the contribution of arachidonic acid (AA) metabolites to the maintenance of cutaneous vasomotor tone after thermal injury, enzyme inhibitors were topically applied to the hamster cheek pouch before and after a spot burn. By use of video microscopy, blood flow was measured in adjacent arterioles that supplied the injured site. Ringer's solutions containing no drug (vehicle), indomethacin (cyclooxygenase inhibitor), BW755c (cyclooxygenase/lipoxygenase inhibitor), or ketoconazole (lipoxygenase/cytochrome P450 inhibitor) continuously suffused the entire tissue. There were no effects of these drugs on preburn blood flow at concentrations that blocked the vascular effects evoked by topical AA. In all groups, blood flow transiently increased after burn and thereafter decreased to levels that were altered by treatment. These results could not be attributed to alterations in vascular reactivity because neither the burn nor the drugs altered the vasodilation evoked by adenosine or prostacyclin. Relative to Ringer's, indomethacin had no effect, BW755c caused vasodilation, and ketoconazole caused vasoconstriction, which suggests that cytochrome P450 products might be vasoactive mediators in injured tissue. Therefore, purified synthetic compounds were compared with known vasodilators. The potency was prostacyclin greater than 12R-hydroxyeicostetraenoic acid greater than adenosine = 5,6 epoxyeicosatrienoic acid greater than AA, which supports the hypothesis that AA can be the source of a novel class of nonprostaglandin vasodilator compounds. In addition, at least one of the vasodilator responses was stereospecific. Nevertheless, the exact explanation for the differential effects of AA inhibitors on postburn blood flow is unknown.

Hamster cheek pouch carcinoma: effect of incision and cortisone on growth, invasion, and metastasis.

After cheek pouch carcinomas were induced in hamsters by the application of dimethylbenzanthracene (DMBA) to the right pouch for 13 weeks, the animals were divided into four groups and observed for seven more weeks. The control group received no further treatment, two experimental groups had incisional biopsies performed on tumors in their pouches, one of these also received injections of cortisone throughout the 20-week experimental period, and a fourth group received cortisone only. The wet weights of the cancerous cheek pouches were determined, and the submandibular and parotid salivary glands with associated cervical lymph nodes, the lungs, and the liver were examined with light microscopy. The cancerous pouches of the animals that received cortisone weighed significantly less than those of animals that received no cortisone but had incisional biopsies of the tumors. There was no significant difference in the degree of histodifferentiation of the tumors among the four groups. The animals in the two groups that received cortisone had significantly more tumors that were invasive than did the animals that did not receive cortisone. Cervical lymph node metastasis occurred in 21% to 38% of the animals but was not significantly different in the four groups. Distant metastases to the lungs or the liver were not found. Incisional biopsy of the tumors stimulated local growth of the cheek pouch tumors, and systemic cortisone administration produced more invasive cheek pouch tumors.

Effects of AVP and DDAVP on histamine-induced increases in macromolecular permeability in the hamster cheek pouch.

The effects of histamine alone and in the presence of AVP or DDAVP on microvascular permeability to macromolecules was evaluated in the superfused hamster cheek pouch. FITC-Dextran (MW 70,000) was employed as a macromolecular tracer to quantitate the increase in macromolecular permeability produced by the topical application of histamine. Intra-vital light microscopy was utilized to quantitate and localize FITC-D extravasation sites along the vascular tree, and fluorimetric measurement of the FITC-D concentration in the suffusate (S) and plasma (P) was used to calculate the FITC-D S/P ratio to quantitate the increase in macromolecular permeability. The infusion of histamine for 5 minutes at a rate which produced a suffusate histamine concentration of 1 X 10(-5) M produced a marked increase in the number of venular FITC-D leakage sites, the [FITC-D]s, and the FITC-D S/P ratio. These effects of histamine were prevented by treatment with either AVP or DDAVP which was infused at a rate sufficient to produce a suffusate concentration of 1 X 10(-8) M. AVP produced profound vasoconstriction whereas DDAVP prevented the histamine-induced increase in the formation of venular FITC-D leakage sites, the [FITC-D]s, and FITC-D S/P ratio without producing vasoconstriction. These data suggest that the antagonism of the histamine-induced increase in macromolecular permeability by AVP and DDAVP is not dependent on vasoconstriction per se, but rather is attributable to the stimulation of a vasopressin receptor on the venular endothelial cell which is identical to or similar to the vasopressin receptor mediating the anti-diuretic effects of these agents.

Effects of chlorhexidine on the structure and permeability of hamster cheek pouch mucosa.

This study examined the effects of chlorhexidine (CHD) on the clinical appearance, morphology, and in vitro permeability of hamster cheek pouch mucosa. The cheek pouches were treated daily for 3 weeks with topical applications of saline, 0.2% CHD, or 2.0% CHD. Treatment with 2.0% CHD resulted in the formation of discrete white lesions in every animal in the group, whereas no changes were identified in any animal treated with 0.2% CHD or saline. Upon microscopic examination it was determined that treatment with 2.0% CHD resulted in a statistically significant (P less than 0.01) increase in epithelial thickness, when compared to the other groups, and the lesions were found to consist of hyperplastic areas of epithelium with associated inflammatory cell accumulations. Daily treatments with 2.0% CHD, 0.2% CHD or saline had no effect on the very low permeability of cheek pouch mucosa to 14C-CHD. However, treatment with 2.0% CHD resulted in decreased permeability to 3H2O (P less than 0.05) when compared to the other groups. Treatment with 2.0% CHD also resulted in a thickened permeability barrier (P less than 0.01), as determined using a tracer, horseradish peroxidase. It is concluded that topical applications of 0.2% T CHD have no detectable effect on cheek-pouch mucosa while applications of 2.0% CHD result in hyperplasia and a decrease in mucosal permeability. Our results suggest that CHD should be used with caution clinically and at a concentration of 0.2% or less.

Antiinflammatory agents inhibit microvascular permeability induced by leukotrienes and by stimulated human neutrophils.

One pathologic change common to the inflammatory process is loss of microvessel membrane integrity which results in edema. Polymorphonuclear leukocytes (PMN) are primary contributors to the development of edema because they cause tissue injury which alters vascular permeability and hemodynamics. The aim of this study was to assess the influence of arachidonic acid metabolites generated by activation of human PMN on the in vivo microvascular preparation of the hamster cheek pouch. Fluorescein-labeled dextran MW: 150,000 was used to assess microvascular permeability. Human PMN were activated with arachidonic acid (AA) and the calcium ionophore A23187, and the supernatant retained for testing. Topical application of the PMN supernatant, purified LTD4 or LTB4 resulted in marked extravasation of macromolecules from post-capillary venules of control hamsters. The extravasation was reduced when hamsters were pretreated with indomethacin (5 mg/kg), imidazole (25 mg/kg), ketoconazole (10 mg/kg), 13-azaprostanoic acid (30 mg/kg), FPL 55712 (1 mg/kg) and dimethylthiourea (500 mg/kg). The interpretation of the results suggests that the increased vascular permeability induced by PMN secretions may be mediated in part by the thromboxane pathway.

The effects of intermittent and continuous stimulation of microvessels in the cheek pouch of hamsters with histamine and bradykinin on the development of venular leaky sites.

Continuous and intermittent stimulation of the hamster cheek pouch with either histamine or bradykinin was studied to evaluate microvascular permeability. Microscopy was accomplished by placing a chamber in the cheek pouch, suffusing the membrane with Ringer's bicarbonate (pH 7.4, 35 degrees C) and observing the microvessels with a Zeiss epistage microscope. Using FITC-dextran (MW 70,000) as the intravascular tracer, the number of leaky sites per cm2 tissue was quantitated before, during and after exposure to histamine (10(-5) M) or bradykinin (10(-7) M). Continuous superfusion with histamine or bradykinin for 60-90 minutes produced an increase in leaky sites which returned toward control during the superfusion. Intermittent stimulation with either histamine or bradykinin produced a maximal response which was not reduced by subsequent 30 minute pulses; whereas reducing the stimulus interval to 15 minutes led to a reduction in leaky sites on subsequent applications. After termination of continuous stimulation with either agent, the application of the other agent produced a response which was similar to the original. There was a minor reduction of responsiveness to intermittent applications of either histamine or bradykinin when the stimulus interval was greater than 15 minutes. These results support the concept of a receptor-mediated modulation of venular leaky sites.

Comparative reactivity of hamster cheek pouch microvessels to arginine vasopressin and angiotensin II.

Experiments were carried out to determine the relative sensitivity of hamster cheek pouch vessels to arginine vasopressin (AVP) and angiotensin II (AII). Hamsters were anesthetized with pentobarbital sodium (6 mg/100 g, ip), a plastic chamber inserted into the cheek pouch and the membrane exposed. The membrane was suffused continuously with bicarbonate-buffered Ringer's solution at 36 degrees C while constantly monitoring blood pressure. After stabilization (30 min) of the membrane, arterioles (30-80 microM diam) were selected for application of AVP or AII in a random fashion. The peptides were applied to the vessels through a micropipette (10-15 mM tip diam) over two minutes using a pump. Total volume delivered was always 20 microliters irrespective of the total amount of peptide (10(0)-10(-4) ng) applied. Vessel diameter was monitored continuously with a shearing device before, during and after the administration of the peptide. The following results were obtained tachyphylaxis was noted to AII but to AVP; the dose response curve for AVP was shifted to the left of that for AII with the threshold dose for AII one hundred times more than that of AVP and AVP had little effect on venules whereas AII produced venoconstriction. These results indicate that AVP is a more potent vasoconstrictor than AII, whereas AII is a more potent venoconstrictor.

Rapid promotion and progression of fibrovascular polyps by inflammation and/or hyperplasia in hamster check pouch: implications for carcinogenesis assay.

Tumor initiation by topical application of 7,12-dimethylbenz[a]anthracene (DMBA) in dimethyl sulfoxide (DMSO) followed by topical application of retinyl acetate (RA), ethylphenylpropiolate, or acetic acid in DMSO at inflammatory and hyperplasiogenic dose regimens caused the rapid promotion of fibrovascular polyps with dysplastic epithelium in hamster cheek pouch. Such lesions did not occur in control animals initiated with DMBA followed by application of DMSO only, where inflammation was also minimal. At the dose regimen employed, RA caused obvious cytotoxicity and tissue destruction. With EPP and AA, there was no histological evidence of tissue destruction. At dose regimens resulting in minimal inflammation and no apparent cytotoxicity, RA promoted almost no polyps, but a higher yield of other tumor types. Thus, inflammation and/or hyperplasia apparently exerted a strong polyp-promoting and progressive influence. This and other differences between the tumorigenic responses of hamster-pouch mucosa and mouse skin suggest that the former supplement the latter in carcinogenic risk assessment.

Enhancement of the sensitivity of hamster check pouch arterioles to beta-adrenergic stimulus during pregnancy.

The adrenergic beta stimulant fenoterol induced a dose-dependent vasodilatation of hamster cheek pouch arterioles. The response to fenoterol was significantly larger on day 14 of pregnancy than in metoestrous animals. Since the serum progesterone and 17 beta-oestradiol level were also elevated on day 14, a relationship was suggested between the enhancement of vascular sensitivity and sex-steroid hormone levels.

Beta-endorphin. Vasodilating effect on the microcirculatory system of hamster cheek pouch.

beta-Endorphin, enkephalins and morphine dilated in vivo the arteriole of the microcirculatory system in hamster cheek pouch. beta-Endorphin had the highest potency and morphine the lowest. The effect was dose dependent and blocked competitively by the opiate antagonist, naloxone. The result demonstrated an effect of the opiates on the arteriole of the cutaneous circulation. This effect probably plays an important role in thermoregulation.

Bradykinin and prostaglandin E1, E2 and F2alpha-induced macromolecular leakage in the hamster cheek pouch.

The hamster cheek pouch prepared for intravital observations on macromolecular permeability with fluorescein labelled dextran was used in four series of 5 hamsters each, all pretreated with indomethacin. Bradykinin, PGE1, PGE2 and PGF2alpha increased macromolecular leakage at postcapillary venules, and this leakage was reversible on removal of agent. A linear relation was found between the logarithmic value of dose of bradykinin and the mean number of leakage sites. No tachyphylaxis to bradykinin was seen. The effect of either PGE1, PGE2 or PGF2alpha applied simultaneously with bradykinin was to significantly (p less than 0.05) potentiate the bradykinin response. Bradykinin and these prostaglandins appeared to have the same site of action for their effect of increasing permeability, e.g. the postcapillary venule.