Time-course of recovery of 5-HT1A receptors and changes in 5-HT1A receptor mRNA after irreversible inactivation with EEDQ.

In this study, the relationship between the expression of 5-HT1A receptors and level of receptor mRNA in discrete regions of rat brain was examined by inactivation of 5-HT1A receptors with the alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ; i.p., 10 mg/kg) and measurement of the time-course of receptor recovery and changes in receptor mRNA levels. Inactivation of 5-HT1A receptors ranged from 84% in the dorsal raphe to 97% in the cortex 12 h after administration of EEDQ. Receptor levels returned to 62-100% of control levels by day 7 and the rate of recovery was uniform across all regions examined. The rate of recovery of 5-HT1A receptors labeled by the agonist [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) and by the putative antagonist [125I]4-(2'-methoxy)phenyl-1-[2'-(N-2"-pyridinyl)-p-iodobenzamido] ethylpiperazine ([125I]p-MPPI) did not differ across regions, suggesting that the ratio of high versus low affinity states of the 5-HT1A receptor remains relatively constant during receptor recovery. However, there did appear to be a short lag in the recovery of sites labeled with the agonist. Significant increases in 5-HT1A receptor mRNA levels were observed as early as 12 h after treatment in all regions but the magnitude of these increases varied. The time-courses of recovery of 5-HT1A receptors and changes in mRNA levels were not parallel in individual regions. Moreover, inactivation of low (8-26%) to moderate (29-57%) levels of 5-HT1A receptors produced no change in mRNA levels, whereas inactivation of greater than 90% elicited a robust increase in mRNA levels. Thus, changes in 5-HT1A receptor expression are not mediated exclusively by changes in mRNA levels and extensive receptor inactivation is required to trigger transcriptional regulation.

Monitoring skin fluorescein delivery independent of skin pigmentation.

This study evaluated two changes in fluorometric assessment of fluorescein dye delivery: (1) monitoring the proportional, rather than the absolute, increase above the preinjection background reading at each site to obviate the effect of skin color and (2) employing gradual means of dye administration to facilitate monitoring uptake kinetics and to lessen the likelihood and severity of histamine-mediated reactions. In 15 beagles, background and postdye readings were obtained with the fiberoptic fluorometer at 27 pairs of matched light- and dark-skin sites of intact perfusion. After dye administration, the absolute increase in fluorescence at light sites averaged more than twice (approximately 225%) that at dark sites. However, the newly introduced proportional increase (PI) was independent of pigmentation. The PI of comparably perfused light and dark sites differed by an average of 3.9% (p greater than 0.05 for difference between PIdark and PIlight). This consistency was maintained after each of three methods of fluorescein administration. After the color independence of the PI was established, its relation to perfusion was assessed in nine 3 X 12 cm pedicle flaps of varied pigmentation. Background fluorometric readings were obtained at eight sites along the longitudinal axis of each flap. Dye was administered as a 20-minute 0.25 mg/kg/min infusion. The PI clearly delineated gradations in perfusion, correlating highly with more time-consuming kinetic indexes and accurately predicting viability. The pedicle row, last viable row (V1), and first nonviable row (N1) had mean PI values of 10.5 +/- 4.5, 4.2 +/- 2.6, and 0.1 +/- 0.1, respectively, at 25 minutes after the start of infusion (p less than 0.05 for pedicle vs V1, pedicle vs N1, and V1 vs N1).

The effects of a topical PGE2 analogue on global flap ischemia in rats.

The present study evaluated the ability of DHV-PGE2ME, a topically effective 16-vinyl prostaglandin E2 analogue, to improve the tolerance of skin flaps to a period of ischemia. DHV-PGE2ME and placebo were applied to bilateral island flaps on 70 anesthetized rats; then the vascular pedicle of each flap was clamped for 10 hours. Treated flaps evidenced significantly better reperfusion, as documented by quantification of fluorescein dye delivery at 90 minutes after clamp release, and they had significantly greater ultimate viability (p less than 0.05, by ANOVA). While less than 3 percent of untreated flaps survived, those treated with 1.75 and 17.5 microgram/cm2 of drug evidenced 76 and 86 percent survival, respectively. Treatment of a given flap did not affect its contralateral mate, since there was no evidence of a systemic effect. Especially since its effect can be limited to the site of application, DHV-PGE2ME should be valuable for the treatment of compromised perfusion in a variety of settings.

Doppler ultrasound, laser Doppler, and perfusion fluorometry in bowel ischemia.

Improved accuracy and objectivity in the evaluation of intestinal viability has been reported by some investigators using Doppler ultrasound, and more recently laser Doppler velocimetry and perfusion fluorometry. To compare the sensitivity and clinical applicability of these techniques, intestinal viability was evaluated by each method in nine 15- to 50-cm loops of small bowel prepared by division of the mesenteric vasculature in five anesthetized dogs. The sensitivity of Doppler ultrasound was 86%, of laser Doppler flow velocity 85%, of laser Doppler index 94%, and of perfusion fluorometry 95%. Though the sensitivity of Doppler ultrasound is significantly less than that of laser Doppler and perfusion fluorometry, this is not unexpected since the latter two techniques are more quantitative than Doppler ultrasound. Clinically, Doppler ultrasound compares favorably with laser Doppler and perfusion fluorometry, and its low cost and simplicity suggest its adjunctive use in the operative setting.

Fluorescence assessment of skin perfusion after oral fluorescein.

This study evaluated oral ingestion as the route of fluorescein administration for fluorescence assessment of skin perfusion. In 10 anesthetized rats, dorsal pedicle flaps were raised to produce graded perfusion; then fluorescein (7.5 mg/kg) was administered via an oropharyngeal tube. Quantification of skin fluorescence, performed with the fiberoptic fluorometer 45 minutes after dye ingestion, delineated a significant difference between the perfusion of flap regions that remained viable and those that subsequently became dystrophic (p less than 0.05). The precision was equivalent to that previously reported after intravenous dye. In addition, the gradual delivery of dye after oral administration permitted monitoring of slope of uptake and time to peak. Each of these pharmacokinetic parameters delineated a significant difference between viable and nonviable sections. As measures of relative change in fluorescence, they are independent of skin color and thickness. This feature suggests that the oral route actually may improve the accuracy and reliability of the fluorescein test.

Fluorometric documentation of increased cutaneous blood flow after topical application of a PGE2 analog in man.

The present study employed fiberoptic fluorometry, a noninvasive means of documenting delivery and removal of fluorescein dye, to evaluate the local circulatory changes elicited by topical application of DHV-PGE2 ME, an investigational PGE2 analog. On Day 1, inactive vehicle was applied to a 5 X 4 cm study site on each thigh of healthy volunteer subjects (n = 12). Symmetrical perfusion was confirmed by similar determinations of dye delivery and removal at each site. On Day 2, DHV-PGE2 ME, 30 or 120 micrograms, was applied to one site while inactive vehicle again was applied to the other. After administration of 120 micrograms in a petrolatum vehicle, fluorometry detected a pronounced increase in nutritive perfusion. There was significant acceleration of dye delivery and removal (p less than 0.05 by ANOVA). Less pronounced changes were noted after the lower dose of DHV-PGE2 ME and when the drug was applied in a triethyl citrate vehicle. The local circulatory changes were not accompanied by systemic effects; there were no changes in vital signs or in fluorometric indices at remote sites.

Fluorometric quantification of low-dose fluorescein delivery to predict amputation site healing.

This retrospective study evaluated quantification of skin fluorescein delivery by fiberoptic fluorometry as a means of predicting the healing potential of an amputation site. Fluorometry uses a dual-channel fiberoptic light guide--one channel transmits blue light to excite the fluorescein in the skin under study, and the other transmits emitted fluorescence from the skin to a photomultiplier tube where it is measured. Ten minutes after intravenous administration of sodium fluorescein (4 to 8 mg/kg), fluorometric readings were obtained at more than 100 reading sites. In the 86 cases without preoperative cellulitis at the site of amputation, preoperative fluorometry clearly distinguished between healing and nonhealing sites. Healing sites averaged 79% of the fluorescence of a healthy reference area (dye fluorescence index [DFI] = 79), while failing sites averaged only 27% (p less than 0.01 by ANOVA). In all but one case where the DFI was greater than 42, the amputation healed. In all cases where the DFI was less than 38, the amputation failed. In general, uncertainty was limited to sites with values between these limits. The technique maintained its high accuracy in patients with diabetes and for distal amputations. However, it was not accurate at sites of active cellulitis (12 cases). There were no significant adverse effects from the slow injection of the low dose of fluorescein used for this technique. We conclude that fluorometry is an effective means of predicting healing in patients undergoing amputation.

Fluorescein elimination as a measure of island flap perfusion.

The fluorescein test has been used as a means of assessing flap viability for almost 40 years. This study was performed in an attempt to determine if recording of the elimination of dye--combined with dye uptake measurement--could provide reliable and consistent monitoring of island flap perfusion.

Intravenous nitroglycerin as a means of improving ischemic tissue hemodynamics and survival.

The effects of intravenous nitroglycerin infusion on experimental skin flap survival and fluorescein dye kinetics were studied in animals. Perfusion fluorometry was utilized to examine changes in flap hemodynamics and viability. An improvement in skin flap survival was found in the nitroglycerin-infused animals, whether the nitroglycerin was initiated 30 minutes before or 30 minutes after flap elevation. Dye elimination in the flap appeared to be significantly improved with the drug therapy, indicating an improvement in flap blood flow with nitroglycerin.

A new technique for evaluating cutaneous vascularity in complicated conjoined twins.

Ischiopagus tripus conjoined twins were recently encountered. All organ systems were thoroughly evaluated using radiographic, angiographic, radionuclide, and CT imaging techniques. None of these studies were capable of accurately assessing the vascular territories of the skin at the level of the pelvis, the most critical area in terms of separation. Qualitative visual assessment of tissue fluorescein delivery under ultraviolet illumination is subject to subjective errors. Perfusion fluorometry is a recently developed technique whereby tissue fluorescence can be quantitated over time in very small amounts, even with repeated injections. In these conjoined twins, using sequential fluorescein injections in each twin, it was possible to accurately determine which portions of the pelvis and the shared leg belonged to which twin and exactly where the skin incisions should be made. Additionally, during operation it was possible to accurately predict the viability of skin flaps used for closure. Both twins survived. This is the first time it has been possible to accurately assess vascular territories of the skin in a complicated form of conjoined twinning. The technique is also useful in the evaluation of flow patterns in various other parts of the body.

Comparative assessment of blood flow to canine island flaps.

Fluorometric quantification of dye delivery, laser Doppler flowmetry, and transcutaneous measurement of tissue oxygen level were evaluated in canine island flaps subjected to vascular occlusion. Each technique readily identified the clamping insult. Relative fluorescein delivery to the flap (compared with a reference area) decreased from 66% before clamping to almost zero during clamping. Laser Doppler readings rapidly declined from an average of 230 to an average of 31 mV. Oxygen readings rapidly declined from 57 to 1 mm Hg. Laser Doppler flowmetry and transcutaneous oxygen monitoring seem best suited for continuous monitoring of trends at a single site. Fluorometry is more precise and can be used to monitor multiple sites. One should weigh the benefits of this technique against the need for dye injection.

Serial fluorometric documentation of fluorescein dye delivery.

Since its introduction in 1980, fiberoptic fluorometry has improved the precision and objectivity of the fluorescein test as a means to delineate nutritive blood flow and predict tissue viability. This study was undertaken primarily to determine if fiberoptic fluorometry could be repeated reliably at close time intervals. Four injections of fluorescein dye were administered at varying dose and time schedules to determine what factors influenced the repeatability of this technique. The results confirm the high accuracy of fluorometric indices as a means to predict the ultimate fate of a skin flap. A method of scanning the flap within 2 minutes after injection of 1.5 mg/kg of fluorescein dye correctly predicted the fate of almost 100% of flap sections. Repeated injections of 1.5, 3.0, and 6.0 mg/kg of dye at 15-minute intervals provided virtually identical predictions of flap viability. Thus fluorometry is readily repeatable and should be of value in assessing the state of tissue perfusion as well as changes in perfusion caused by pharmacologic manipulation or surgical intervention.

Fluorometric prediction of successful amputation level in the ischemic limb.

The present study was undertaken to compare fluorometric documentation of fluorescein dye delivery with the standard means of determining the level at which an amputation should be performed in the dysvascular extremity. Thirty-nine patients underwent lower-extremity amputation at the level determined by the surgeon based upon physical examination, angiography, segmental pressure indices, and/or pulse volume recordings. In addition, fiberoptic fluorometry was performed preoperatively. After intravenous administration of sodium fluorescein (4-8 mg/kg), fluorometric readings were obtained by placing the fluorometer's light guide on 126 reading sites. Fluorometric findings were evaluated retrospectively, and therefore did not influence the surgeon's decision. Of the 39 amputations performed overall, only 26 healed. The accuracy of the standard criteria was lowest for the 20 below-ankle amputations, where only 12 cases healed. Alternatively, fluorometric indices separated healing from nonhealing sites in 36 of the 39 cases and in 18 of the 20 below-ankle amputations. Overall, healing sites averaged 94 percent of the fluorescence of the healthy reference area, while nonhealing sites averaged only 29 percent. We conclude that fluorometry should prove to be a valuable adjunct in the assessment of the dysvascular extremity. It uses a low dose of dye, is easy to perform, and is readily repeatable.

Assessment of delay in bilateral thoracoabdominal island flaps in the rat.

This study demonstrates improved perfusion and viability following surgical delay in a bilateral paired flap model that minimizes the effects of systemic variables in the rat. Surgical delay was achieved in one of two paired thoracoabdominal island flaps by ligating and cutting the inferior epigastric (distal) pedicle three days before flap construction. This created a hypoperfused zone. Within three days of delay, perfusion of the delayed region, as documented by quantification of tissue fluorescein delivery, increased markedly and often exceeded nondelayed values. Construction of 2 X 11-cm island flaps based on the superior epigastric pedicle at this time was associated with an average of 1.1-cm necrosis, compared with 2.6 cm in nondelayed flaps.

Vascular island skin-flap tolerance to warm ischemia: an analysis by perfusion fluorometry.

Fiberoptic perfusion fluorometry and assessment of ultimate viability were used to analyze the tolerance to warm ischemia of rat vascular island skin flaps. Both acute flaps and flaps raised 24 hours previously and then reraised were subjected to 0 (control), 6, 8, 10, and 12 hours of vascular pedicle clamping. Following clamp release, serial fluorometry documented the progressive delay in effective reflow resulting from extended periods of ischemia. Fluorometry, furthermore , suggested that flaps constructed 24 hours previously had an improved hemodynamic status with a significantly reduced period of poor reflow following clamp release. The improved hemodynamics were associated with increased viability, confirming the increased tolerance of 24-hour-old flaps to warm ischemia.

Assessment of neovascularization and timing of flap division.

These studies were undertaken to determine if perfusion fluorometry can provide an accurate, reliable means of monitoring neovascularization and predicting optimal time of flap division. An animal model was developed that permitted daily assessment of neovascularization of the flap using fluorescein injections and the fluorometer. An increase in fluorescence of the pedicle-occluded flap of approximately 12 percent the normal skin fluorescence was associated with 100 percent flap survival. This occurred after only 4 days in the rat. Fluorometry proved valuable in the evaluation of clinical flaps. The fluorescence of flaps during pedicle occlusion was observed to increase with each successive fluorometric evaluation performed at 3- to 7-day intervals. It appears that flaps after pedicle occlusion exhibiting at least 25 percent of the fluorescence of normal skin will tolerate pedicle division. Based on the neovascularization studies of the clinical flaps, all pedicles were severed earlier than originally anticipated. Two cross-leg flaps were successfully divided after 11 days. Quantitative assessment of serial fluorescein injections allows reliable evaluation of neovascularization in clinical interpolation flaps and is useful in the timing of flap division.

Fluorometric assessment of skin flap viability in the rat: effect of radiation therapy.

The fluorescein test is widely used to assess perfusion in skin flaps but tends to underestimate skin viability when visual inspection under ultraviolet light is employed. Fiberoptic dermofluorometry, which has recently been introduced, more accurately assesses fluorescein distribution in skin flaps than does visual inspection. Viability of the back flap in rats receiving ionizing radiation was evaluated by dermofluorometry. This technique was highly accurate in predicting viability and has great applicability for studying blood flow in irradiated tissues.

Quantification of fluorescein distribution to strangulated rat ileum.

Following various periods of strangulation, the fates of intestinal segments were predicted by standard clinical criteria and visual (Wood's lamp) and fluorometric (perfusion fluorometer) assessment of fluorescein distribution. With fluorometry, a means of quantifying fluorescence transmitted via a fiberoptic light guide, the delivery and removal of fluorescein were monitored and analyzed. If either was restricted significantly, tissue death was predicted. Analysis of computerized graphic patterns or simple interpretation of fluorometric readings at two time points predicted tissue fate with 98% accuracy and a 93% negative predictive value. Wood's lamp evaluation had only a 53% accuracy and a 33% negative predictive value, while standard clinical criteria had an 81% accuracy and a 53% negative predictive value. Fluorescein leakage in segments which suffered significant endothelial damage provided staining patterns that incorrectly suggested viability. By monitoring elimination as well as uptake of dye, fluorometry provided much greater discrimination than did Wood's lamp inspection in this setting. In addition, fluorometry was readily repeatable within minutes, as fluorescence remaining from a previous injection could be subtracted from new, postinjection values.