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Journal of Pediatric Surgery (2008) 43, 1672–1678

Responses of the murine esophageal microcirculation toacute exposure to alkali, acid, or hypochlorite☆

Mohammad Osmana,c, Janice Russella, Deepty Shuklab,Mana Moghadamfalahib, D. Neil Grangera,⁎

aDepartment of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center,Shreveport, LA 71103-3932, USAbDepartment of Pathology, LSU Health Science Center, Shreveport, LA 71103, USAcDepartment of Pediatric Surgery, Ain Shams University, Cairo, Egypt

Received 23 October 2007; revised 20 January 2008; accepted 22 January 2008

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Key words:Esophagus;Caustic injury;Intravital microscopy;Leukocyte adhesion

AbstractBackground/Purpose: Although ingestion of alkali-based and/or hypochlorite-based household cleanersas well as strong acids remains a major cause of esophageal wall injury, little is known about themechanisms that underlie the injury response to these toxic agents. This study examined the roles ofvascular dysfunction and inflammation to the esophageal injury response to different caustic substancesin mice.Methods: The esophageal responses to sodium hydroxide (10%, 5%, and 2.5%), potassium hydroxide(10%, 5%, and 2.5%), sodium hypochlorite (5.25%), and hydrochloric acid (10%, pH 2) were evaluatedby intravital videomicroscopy and histopathology. Intravital microscopy was used to monitor changes inthe diameter of arterioles and venules, the adhesion and movement of leukocytes in venules, and thetime of cessation of arteriolar blood flow in mouse esophagus. The esophageal mucosa was exposed tocaustic substances for 0 to 60 minutes before evaluation.Results: The higher concentrations of sodium hydroxide and potassium hydroxide elicited rapid stasisin both arterioles and venules, which was accompanied by arteriolar constriction and thrombosis. Anaccumulation of adherent leukocytes in venules was not observed with any agent. Histopathologicalevaluation revealed marked cellular and interstitial edema in the mucosa with alkali, whereashydrochloric acid and sodium hypochlorite decreased the thickness epithelial layer.Conclusion: These findings suggest that ischemia and thrombosis are dominant processes, whereasinflammation is less important in the pathogenesis of acute corrosive injury to the esophageal mucosa.© 2008 Elsevier Inc. All rights reserved.

Ingestion of caustic substances remains a common health

☆ This study was supported by a grant (DK43875) from the Nationalstitute of Diabetes and Digestive and Kidney Diseases.⁎ Corresponding author.E-mail address: dgrang@lsuhsc.edu (D.N. Granger).

022-3468/$ – see front matter © 2008 Elsevier Inc. All rights reserved.oi:10.1016/j.jpedsurg.2008.01.069

problem that affects children worldwide and continues torepresent the most common cause of esophageal stricture inthis age group [1]. Medical problems caused by causticingestion are associated with both acute and chroniccomplications. Acute complications include local burn and

1673Microvascular responses to esophageal injury

interstitial edema in upper gastrointestinal passages andaspiration with subsequent respiratory complications.Chronic complications include esophageal stricture, malnu-trition, and psychological problems [2].

Previous experimental studies of caustic ingestion havelargely focused on potential therapeutic agents/interventionsthat either antagonize the acute effects of the causticsubstance or reduce the deposition of fibrous tissue andsubsequent stenosis that often follows the injury. Althoughcorticosteroids and antibiotics have been advocated to reducethe inflammatory response initiated by caustic ingestion, theclinical benefits of these treatments remain controversial[3-17]. Most drug therapies for caustic ingestion that havebeen tested to date have not shown significant clinicalefficacy, and endoscopic dilatation or surgical resectionremains an option for management of patients with severecomplications resulting from caustic ingestion [2,7,15,16].

The search for effective treatments for esophageal injurythat result from caustic ingestion has been hampered by aclear understanding of the nature, time course, and under-lying mechanisms of the tissue responses to caustic agents.Strong acids are believed to cause necrosis throughdenaturation of proteins (coagulation necrosis), whereasstrong alkalis mediate necrosis via the saponification of fatsand solubilization of proteins (liquefactive necrosis) [2].However, little else is known about the pathophysiology ofcaustic injury to the esophagus [1]. Although thrombosis,ischemia, and inflammation have all been implicated in thepathogenesis of caustic injury, there is no direct evidenceshowing the genesis of these responses in the esophagealmucosa after exposure to strong acids or alkali.

Intravital microscopy (IVM) is a widely used method tovisualize and study the microcirculation in living tissue. Thistechnology allows for quantification of different physiologi-cal variables, including vessel diameter, perfusion rate,pressures, and permeability. The application of specificfluorochromes and fluorescence imaging with IVM alsoenables the user to quantify the movement of specific bloodcell populations within the vessel lumen as well as theadhesive interactions between these cells and vessel wall[18]. In the present study, IVM was used to monitor changesin the diameter of arterioles and venules, the adhesion andmovement of leukocytes in venules, and the time of cessationof arteriolar blood flow in mouse esophagus after exposure todifferent caustic agents. The overall objective of this studywas to determine whether vascular dysfunction and inflam-mation are early components of the esophageal injuryresponse to commonly ingested caustic substances.

1. Materials and methods

1.1. Animals

Male C57BL/6J mice (n = 121) were purchased fromThe Jackson Laboratory (Bar Harbor, ME, USA). Animals

were maintained on a 12-hour light/12-hour dark cycleunder pathogen-free conditions. The mice had ad libitumaccess to a standard diet and water until reaching the desiredage (8-10 weeks) and/or weight (20-25 g). All proceduresusing animals were reviewed and approved by theInstitutional Animal Care and Use Committee of LouisianaState University Health Sciences Center and were per-formed according to the criteria outlined by the NationalInstitutes of Health.

1.2. Caustic substances

The following substances were evaluated: sodiumhydroxide (NaOH; SigmaUltra; pellets, minimum concen-tration 98%) diluted into 3 concentrations: 10%, 5%, 2.5%;potassium hydroxide (KOH; SigmaUltra; pellets, KOH≥85%, K2CO3 ≤2.0%) diluted into 3 concentrations: 10%,5%, 2.5%; Sodium hypochlorite (NaOCl) 5.25% (commer-cial Clorox); hydrochloric acid (HCl) diluted into 2concentrations, 10% and pH 2. Saline was used as thecontrol solution.

1.3. Intravital microscopy experiments

Mice (n = 61) were anesthetized subcutaneously with amixture of ketamine and xylazine at doses of 100 and 5 mg/kg, respectively. The right femoral artery and left femoralvein were cannulated with polyethylene tubing (PE-10). Amidline abdominal incision was made, and the lower part ofthe esophagus was cannulated using polyethylene tubing(PE-90) just above the gastroesphageal junction. The tubingwas secured in place with a silk tie. The neck was openedthrough an inverted T incision and the trachea exposed. Theleft sternomastoid and strap muscles of the neck on bothsides were divided by heat cautery to enhance exposure ofthe trachea, which was cannulated PE90 tubing (PE-90)secured in place by silk suture. Traction was applied to thesilk suture to tilt the trachea slightly to the right to expose theunderlying esophagus. Fascia covering the esophagus wasdissected, taking care not to injure the esophageal vascularsupply. A PE-90 cannula for delivery of caustic substanceswas advanced through the mouth to the lower border of thelarynx. A thin strip of Saran wrap was placed over theesophagus to prevent dryness.

The mouse esophageal microcirculation was visualizedusing ×40 water immersion lens of an upright Nikonfluorescence microscope (Nikon Inc, Tokyo, Japan) equippedwith a 75-watt XBO xenon lamp. A solution of 0.2 mL ofrhodamine-6G was injected through the venous cannula andthen the mouse was allowed to stabilize for a period of30 minutes before collection of video images of themicrovasculature. An arteriole and venule within the wallof the esophagus were selected for study. Digital videorecordings of 30-second duration were obtained under control(preexposure to caustic agent or saline) conditions, at 2.5-minute intervals during the first 10 minutes of chemical

Table 1 Effects of different caustic solutions on the time forblood flow cessation in esophageal arterioles and venules

Substance andconcentration

Time to bloodflow cessation (min)

Saline N60HCl 10% N60HCl pH 2 N60Na hypochlorite 5.25% N60Na hydroxide 10% 8.25 ± 1.25 ⁎

Na Hydroxide 5% 16.4 ± 2.8 ⁎ ,†

Na hydroxide 2.5% N60K hydroxide 10% 5.8 ± 0.56 ⁎

K hydroxide 5% 15.3 ± 1.63 ⁎ ,†

K hydroxide 2.5% N60

⁎ P b .05 vs saline, NaOH 2.5%, KOH 2.5%.† P b .05 vs NaOH 10%, KOH 10%.

1674 M. Osman et al.

exposure period and at 5-minute intervals thereafter, until thecessation of blood flow or 60 minutes, whichever occurredfirst. The following variables where measured off-line in eachof the video recordings: internal diameters of the arteriole andvenule, number of leukocytes firmly adhering (stationary) invenules for at least 30 seconds, flux (number per 30 seconds)of rolling leukocytes, time to cessation of blood flow in boththe arteriole and venule. Blood pressure was also monitoredvia the femoral artery cannula.

1.4. Histopathology

Following intraluminal placement of the caustic solution(0.2mL) for a period of 10minutes, the lumenwas rinsedwith1.0 mL of saline. The cervical esophagus was then harvestedand immediately fixed in formalin. The tissue samples werecut into sections, stained with hematoxylin and eosin andevaluated histologically. Quantification of the histologicalchanges was performed using a previously described scoringsystem with a few modifications [5]. The slides were gradedblindly by a pathologist using the following criteria:

Esophageal viability (0—normal esophagus; 1—only mucosalnecrosis; 2—necrosis involving mucosa and superficial musclelayer; 3—necrosis involving all 3 layers: mucosa, superficialmuscular layer, and deep muscle with adventitial layer).– Cornified epithelial cell differentiation (0—present, 1—not present).– Epithelial cell nucleoli (0—normal, 1—nucleoli present onlyin superficial one third of mucosal cells, 2—nucleoli presentonly in superficial two thirds of mucosal cells, 3—nucleolipresent in full thickness of the mucosal cells).– Edema and spongiosis (0—normal, 1—edema and spongiosispresent only in superficial one third of squamous mucosa, 2—edema and spongiosis present only in superficial two thirds ofsquamous mucosa, 3—edema and spongiosis present in fullthickness of the squamous mucosa).– Thrombosis (0—not present, 1—present).– A total histopathology score, with a maximum potentialvalue of 10, was derived from the sum of the individual scoresfor tissue viability, cornified epithelial cell differentiation,epithelial cell nucleoli, edema/spongiosis, and thrombosis.

1.5. Data analysis

Statistical analyses of the data were performed withStatView 4.5 software (Abacus Concepts Inc, Berkeley, CA)using 1-way analysis of variance with Fisher's (post hoc)test. All values are reported as means ± SEM. Statisticalsignificance was set at P b .05.

2. Results

2.1. Intravital microscopy

Blood flows in esophageal arterioles and venules weremonitored for a period of 60 minutes after placement of

saline or different caustic solutions in the esophageal lumen.Table 1 summarizes the times required for blood flowcessation in both arterioles and venules after exposure to thedifferent solutions. Saline, as well as HCl (10% and pH 2.0),NaHOCl (5.25%), NaOH (2.5%), and KOH (2.5%), did notresult in microvascular flow cessation over the 60-minuteobservation period. However, 10% solutions of either NaOHor KOH lead to a very rapid (b10 minutes) and completecessation of arteriolar and venular blood flows. The 5%solutions of the same agents also resulted in blood flowcessation, although a slightly longer time (≥15 minutes) wasrequired to mediate this response.

The responses of arteriolar and venular diameter to thedifferent caustic solutions were also monitored. The resting(control) diameter of arterioles in these experiments was24.2 ± 0.86 μm, whereas the resting venular diameter was44.5 ± 1.4 μm. Arteriolar diameter was largely unaffectedby luminal perfusion with most the caustic agents, withthe exception of HCl (pH 2.0 and 10%) (Fig. 1A). Moreprofound changes in arteriolar diameter were noted with5% and 10% NaOH and 5% and 10% KOH (Fig. 1B),which produced 38%, 18%, 30%, and 27% peakreductions in arteriolar diameter, respectively. Only 2solutions were noted to significantly alter the diameter ofesophageal venules by more than 20% (Fig. 1D), that is,5% KOH and 5% NaOH, which reduced vessel diameterby 49% and 39%, respectively. Ten percent NaOHproduced an 18% reduction in venular diameter, whereasan even smaller (11%-14% reduction) change was notedwith 10% HCl (Fig. 1C).

Under basal conditions, the number of adherent leuko-cytes in esophageal venules (no leukocyte adherence wasnoted in arterioles) was 301 ± 58 cells per mm2 vessel area,whereas the leukocyte flux was 754 ± 82 cells per 30 secondsper mm2. Neither the number of adherent leukocytes norleukocyte flux was significantly altered by exposure of theesophagus to saline or any of the caustic solutions, although

Fig. 1 Changes in esophageal arteriolar diameter over a 60-minute period after intralumenal exposure to (A) NaOCl, HCl (pH 2 and 10%),2.5% KOH, 2.5% KOH, or saline. Panel (B) summarizes the changes for 5% and 10% NaOH and 5% and 10% KOH. With the latter solutions,arteriolar diameter was not measured after flow stasis was detected. Changes in the venular diameter where shown in panel (C) for NaOCl, HCl(pH 2 and 10%), 2.5% KOH, 2.5% KOH, or saline, and in panel (D) for 5% and 10% NaOH and 5% and 10% KOH. #P b .05 compared withcorresponding control value.

1675Microvascular responses to esophageal injury

there was a tendency for a decline in leukocyte recruitmentthat paralleled the changes in venular blood flow.

2.2. Histopathology

A summary of the scoring of histopathological changes inthe esophagus elicited by the different caustic solutions isprovided in (Fig. 2). The total score (sum of individualindices) and the contributions of tissue viability, cornifiedepithelial cell differentiation, epithelial cell nucleoli, edema/spongiosis, and thrombosis to the total score are presentedfor each solution. Although no significant tissue pathologywas evident after exposure of the mouse esophagus to eithersaline, HCl (10% and pH 2), or 2.5% KOH, significanthistological changes were noted with all other solutions.NaOCl elicited a small (1.7 ± 0.6) but significant increase inhistopathology score that reflected changes in epithelialviability, edema/spongiosis, and the presence of a cornifiedlayer as well as thrombosis. The histopathological changes

noted with 5% and 10% KOH, and 2.5%, 5%, and 10%NaOH were substantially larger, with total histopathologyscores ranging between approximately7.5 (5% and 10%KOH) and 8.0-8.6 (2.5%, 5%, and 10% NaOH). Edema/Spongiosis and the appearance of epithelial nucleoli largelyaccounted for the high total histopathology scores inducedby these solutions.

Fig. 3 illustrates representative histopathological changesobserved in the murine esophagus exposed to 10% KOH(Fig. 3B) or 10% NaOH (Fig. 3C) and compares thesechanges to the saline-exposed esophagus (Fig. 3A). Thesechanges are typical of the quantitative histopathologicaldata summarized in Fig. 2 for the same caustic agents andconcentration. Fig. 4 illustrates representative responses ofarterioles and venules in the esophageal microcirculationafter exposure to KOH 10%. Fig. 4A shows both anarteriole and venule before exposure to the caustic agent.Fig. 4B is an image obtained 4 minutes postexposure andshows a marked reduction in the arteriolar diameter withalmost no change in venular diameter. The number of

Fig. 2 Histopathology score for murine esophagus exposed to different caustic solutions. The contributions of thrombosis, edema/spongiosis, cornified layer, epithelial viability, and epithelial nucleoli to the total score are also depicted.

1676 M. Osman et al.

adherent leukocytes in the venule was largely unaffected byKOH exposure.

3. Discussion

The accidental ingestion of caustic agents remains aleading cause of esophageal injury in children worldwide [1].Although this has led to an intensive effort to identifytherapeutic interventions that protect against chemical burnsto the esophagus, no effective treatments for the acute phaseof injury have been found to date. Advancements in this fieldhowever may be limited by the lack of information on howstrong alkali and acids produce rapid tissue necrosis in theesophagus. Although thrombosis, ischemia, and inflamma-tion have all been implicated in caustic injury to theesophagus [1], there are no reports in the literature thatdirectly address the responses of the esophageal micro-vasculature to the injury-inducing caustic agents. In thisstudy, intravital videomicroscopy was used to monitor theearly responses of the murine esophageal microcirculation tonoxious concentrations of alkali and acids to determinewhether these agents elicit changes that are consistent withthrombosis, ischemia, and inflammation. Our findings areconsistent with rapid vascular stasis caused by both vesselconstriction and thrombosis. Our findings also indicate thatthe vascular stasis is not accompanied by the recruitment ofinflammatory cells in the early period following exposure tothe noxious agents.

Previous studies of caustic tissue injury to the esophagushave largely involved the use of rats, rabbits, or largeranimals [12-14]. The present study represents the first effort

to evaluate the toxic effects of strong alkalis and acids inmouse esophagus. The histopathological changes elicited byalkali and acid in mouse esophagus are qualitatively similarto those previously reported for other species and areconsistent with the view that strong acids and alkali causerapid tissue necrosis, edema, and thrombosis in this tissue[5]. Quantitative comparisons of our histopathologicalchanges with those reported in other tissues are difficultbecause different concentrations of the noxious agents andpathologic scoring systems were used. Nonetheless, themurine model for study of esophageal injury followingexposure to caustic agents holds much promise because theanimals are readily available and inexpensive. Moreimportantly, a large number and wide variety of mutantmice are available for detailed analysis of molecularmechanisms that contribute to the pathogenesis of esopha-geal injury. These mutants provide an entirely new strategyfor identifying potential therapeutic strategies for treatmentof burn injuries in the esophagus.

A striking response of the esophageal circulation tocaustic injury was the complete and rapid cessation of bloodflow in both arterioles and venules, which supports the viewthat ischemia is an early manifestation of the injury responseto noxious agents. The highest concentrations of alkali (10%KOH and NaOH) studied led to flow cessation within 5 to 8minutes, whereas 5% solutions of the same agents causedflow cessation within 15 to 16 minutes (Table 1). Ourarteriolar diameter and histopathology data suggest that boththrombosis and vasoconstriction may contribute to the rapidstasis in esophageal arterioles and venules. Exposure of theesophagus to the alkali solutions was usually accompaniedby thrombosis (Fig. 1). Some solutions, such as 5% NaOH

Fig. 3 Representative histopathological changes observed in themurine esophagus exposed to 10% KOH (B) or 10% NaOH (C) andcompares these changes to the saline-exposed esophagus (A).

ig. 4 Typical changes in an esophageal arteriole (A) and venule) after exposure to KOH 10%. Panel (A) shows both an arteriole

nd venule before the exposure. Panel (B) is an image obtainedminutes postexposure.

1677Microvascular responses to esophageal injury

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and 5% KOH, were also associated with significant peakreductions in arteriolar diameter, ranging between 25% and40%. Assuming a cylindrical geometry, arteriolar constric-tion of this magnitude may result in blood reductionsranging between 70% and 90%. The added response ofvenoconstriction that was noted for 5% KOH may alsocontribute to the rapid decline in blood flow. It is likely thatthe combination of vasoconstriction and thrombosis isneeded to elicit the stasis that was observed with the alkalisolutions. Support for this contention is derived from theobservation that HCl (pH 2) was associated with a 23%reduction in arteriolar diameter but less frequently withthrombosis. Similarly, HCl did not lead to flow cessationwithin 60 minutes, nor did it produce significant histologicalchanges. The absence of histological changes with HCl mayreflect the short duration of acid exposure.

Another objective of this study was to determine whetherthe esophageal microcirculation contributes to the early

1678 M. Osman et al.

injury response to caustic agents by mediating the recruit-ment of inflammatory cells. By monitoring the trafficking ofleukocytes in esophageal venules using IVM, we did notobserve a significant increase in the recruitment ofleukocytes within the first hour after caustic injury. Although60 minutes may have been too early to expect significantleukocyte recruitment, previous work in other vascular bedsof the digestive system have revealed large and highlysignificant increases in the number of adherent leukocyteswhen venular blood flow (shear rate) is reduced [19,20]. Thisshear rate-dependent recruitment of adherent leukocytes ismanifested when blood flow is reduced either locally byvessel occlusion or systemically by graded hemorrhage[19,20]. The absence of leukocyte recruitment in esophagealvenules in the face of profound reductions in blood flow istherefore unexpected, but it may reflect the uniquecircumstances (such as thrombosis and direct chemicalinteractions with blood elements) that accompany causticinjury. Our observations in the first 60 minutes afteresophageal exposure to strong alkali and NaOCl do notnegate a role for inflammatory cell infiltration and leukocyte-mediated tissue damage at later time-points. It is possible thatvenular blood flow is eventually restored in some esophagealvenules after caustic injury, allowing for the delivery ofinflammatory cells that have been shown histologically toinfiltrate the esophagus days after exposure to the causticagent [21]. Additional work is needed to address how theinjured esophageal microvessels can sustain this delivery ofinflammatory cells in the face of blood flow stasis.

In conclusion, the results of this study provide novelinsights into the early vascular responses that accompany theesophageal injury induced by strong acid and alkali. Ourfindings indicate that the most toxic noxious agents are alkalithat elicit a rapid cessation of blood flow in esophagealmicrovessels. Vasoconstriction and thrombosis may bothcontribute to the esophageal ischemia induced by alkali. Thechanges in esophageal blood flow are not associated with anenhanced recruitment of inflammatory cells that is mediatedthrough adhesive interactions with venular endothelium.Intravital microscopic observation of the murine esophagealmicrocirculation represents a powerful new approach todefining the mechanisms that underlie the burn injury that isassociated with ingestion of caustic agents.

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