Eric R. Lacy

Rapid epithelial restitution of human and rabbit colonic mucosa

Rapid epithelial restitution is now considered one of the primary defense mechanisms of the stomach and duodenum. Because there is currently no evidence as to whether restitution occurs in human tissue, this study examined human and rabbit colonic mucosa after superficial injury and monitored the potential difference, alkaline flux, and speed and mechanisms of mucosal restitution as observed with light and electron microscopy. Luminal exposure of the in vivo rabbit colon to 100 mM HCl for 5 min or the in vitro human colon to 10 mM HCl for 10 min caused superficial mucosal injury to 76% of the epithelial surface in the rabbit and 95% in the human. The necrotic epithelial cells detached in sheets from the intact basal lamina and formed a protective mucoid layer. Morphologic evidence of restitution occurred within 15 min after injury in the rabbit and 30 min in the human, as viable nongoblet cells projected lamellipodia and migrated over the denuded basal lamina at a speed of approximately 2 microns/min. One hour after damage 61% of the mucosal surface was still damaged in the rabbit, and 86% of the human mucosal surface was damaged after 2 h. In the following 60 min restitution progressed rapidly, so that only 10% of the surface remained unrepaired in the rabbit after 2 h and 19% in the human after 3 h. Small areas with deeper injury did not repair until 5 h after damage. The potential difference dropped after mucosal injury and did not recover despite morphologic repair. Rapid epithelial restitution is considered to be a basic defense mechanism of the gastrointestinal mucosa that is obviously not necessarily related to the presence of an acidic environment in the stomach or duodenum.

Potential Indicators of Stress Response Identified by Expressed Sequence Tag Analysis of Hemocytes and Embryos from the American Oyster, Crassostrea virginica

Abstract: A pilot program was initiated to identify genes from the American oyster, Crassostrea virginica, that are potentially involved in the stress response for use as bioindicators of exposure to environmental pollutants and to toxic and infectious agents. A PCR-based method was used to construct cDNA libraries from pooled embryos and the hemocytes of a single individual. A total of 998 randomly selected clones (expressed sequence tags, ESTs) were sequenced. Approximately 40% of the ESTs are novel sequences. Several potential biomarkers identified include an antimicrobial peptide, recognition molecules (lectin receptors), proteinases and proteinase inhibitors, and a novel metallothionein. Diversity analysis shows that 363 and 286 unique genes were identified from the hemocyte and embryo libraries, respectively, indicating that full-scale EST collection is a valuable approach for the discovery of new genes of potential significance in the molluscan stress response.

Substance P attenuates gastric mucosal hyperemia after stimulation of sensory neurons in the rat stomach

BACKGROUND/AIMS Sensory neurons in the stomach mucosa are closely apposed to mast cells and blood vessels. Mucosal hyperemia, after exposure to capsaicin, is mediated by calcitonin gene-related peptide (CGRP) from these neurons, which also contain substance P (SP). However, the role of this peptide in blood flow regulation remains unclear. Therefore, this study examines the effect of SP on capsaicin-induced mucosal hyperemia and mast cells. METHODS Gastric mucosal blood flow was measured with laser Doppler flow velocimetry in chambered rat stomachs. SP, aprotinin (serine protease inhibitor), and ketotifen (mast cell stabilizer) were infused into the splenic artery of rats. Mast cells were counted by microscopy. RESULTS Mucosal exposure to capsaicin (640 mumol/L) evoked a 70% increase in mucosal blood flow, which was abolished by SP, whereas aprotinin infused with SP and pretreatment with ketotifen before SP infusion restored the hyperemic response. Morphometry showed that ketotifen inhibited mast cell degranulation in SP-treated animals. Preservation of mast cells in SP-treated rats was linearly correlated to increased mucosal blood flow after exposure to capsaicin. CONCLUSIONS These data suggest that SP participates in regulation of gastric mucosal blood flow by activation of mast cells most likely by releasing proteases from mast cells that cleave and inactivate CGRP.

Importance of an alkaline microenvironment for rapid restitution of the rabbit duodenal mucosa in vitro

Rapid epithelial restitution after superficial damage of the gastroduodenal mucosa consists of the migration of remaining intact epithelial cells beneath a necrotic layer of mucus and shed cells. Complete reepithelialization occurs within 60 min (rat stomach) to 7 h (rabbit duodenum) and does not involve cell division. The present study investigated rapid restitution of the acid-damaged rabbit duodenal mucosa in vitro under various conditions. Alkaline flux and transmucosal potential difference were measured simultaneously, and computerized morphometry was performed. Rapid restitution was nearly completed 5 h after damage at neutral luminal pH, but it was retarded when the luminal pH (pHL) was kept at 3. Removal of the necrotic layer did not impair restitution at neutral luminal pH but caused delay at acidic pH (pHL = 3.0). Removal of nutrient bicarbonate slightly delayed restitution at pHL = 7.4 and caused complete inhibition at pHL = 3.0. Alkaline secretion was usually stable but was nearly totally abolished after removal of nutrient bicarbonate. The potential difference decreased after acid damage and showed a tendency to recover in parallel with mucosal restitution. This gradual recovery correlated directly with morphometry. The authors assume that rapid restitution of the duodenal mucosa at an acidic luminal pH depends on the presence of a protective necrotic layer and sufficient alkaline secretion to maintain an optimal environment adjacent to the mucosa. Disturbance of this alkaline microenvironment could be important for the development and healing of duodenal ulcer.

The elasmobranch kidney. I. Gross anatomy and general distribution of the nephrons.

SummaryThe morphology of the little skate (Raja erinacea) and spiny dogfish shark (Squalus acanthias) nephron has been investigated in sexually mature females by 1) gross observations of the kidney surfaces, 2) vascular injections, 3) scanning electron microscopy, 4) light microscopy.In the little skate, each nephron is highly complex and begins at the urinary pole of the renal corpuscle, which is located between a thin, dorsalbundle zone and a thicker, ventralsinus zone. The nephron loops back and forth, repeatedly entering and exiting each zone. In thebundle zone, segments from each nephron form a bundle of 5 tubules (tubular bundle) which are arranged in a countercurrent loop fashion. A peritubular sheath composed of closely packed, squamous cells wraps the 5 nephron segments of the tubular bundle together and separates each bundle from the next. In thesnus zone the tubules from many nephrons mix freely with each other in large blood sinuses.In the spiny dogfish, the nephron displays a complex pattern similar to that of the skate. Renal corpuscles are adjacent to abundle zone composed of tubular bundles, each wrapped by a peritubular sheath in a cell-rich connective tissue matrix. However, thebundle zone is not limited to the dorsal region of the shark kidney but extends ventrally along deep interlobular septa. Thesinus zone of the shark is like that of the skate except that it is not limited to the ventral regions of the kidney.

Role of sensory afferent neurons in hypertonic damage and restitution of the rat gastric mucosa.

BACKGROUND & AIMS Gastric mucosal hyperemia is a protective response mediated at least in part by the response of sensory afferent neurons to hydrogen ions. The aim of this study was to determine if other pathways to the hyperemic response are present and if these neurons have an effect exclusive of hyperemia on mucosal protection and repair. METHODS Rat sensory afferent neurons were ablated by capsaicin treatment. Chambered stomachs were damaged by hypertonic saline followed by either acidic or neutral isotonic saline. Blood flow was measured by laser Doppler velocimetry, and mucosal morphology was quantitatively evaluated by microscopy. RESULTS Mucosal damage alone evoked a strong hyperemic response in both control and ablated rats. Ablated rats lost gastric protection despite this hyperemic response. Acid exposure after damage sustained the hyperemic response. Rapid epithelial restitution occurred faster (even over hemorrhagic lesions) in control rats. CONCLUSIONS The hyperemic response to mucosal damage alone is not mediated by sensory neurons. Protection of the stomach by sensory afferent neurons occurs by mechanisms also unrelated to their elicitation of hyperemia. Restitution during acid challenge is enhanced by the sustained hyperemic response mediated through sensory afferent neurons.

Rapid repair of the surface epithelium in human gastric mucosa after acute superficial injury.

Morphologic changes associated with rapid epithelial repair in human gastric mucosa were examined by light and transmission electron microscopy in samples obtained at biopsy. A timed series of samples covering six intervals (3-30 min) after exposure to 50% (vol/vol) ethanol was available. Samples exposed to 25% ethanol and to acidified sodium taurocholate were also studied. Changes indicative of epithelial repair were first seen in samples fixed 15 min after ethanol. In samples fixed 30 and 45 min after exposure to barrier breakers, there were widespread changes indicative of epithelial repair. We have tentatively identified three morphologically distinct types of repair: lateral movement of the epithelial cell basal plasma membrane, which maintains close contact with the basal lamina and which may occur only in the presence of continued vascular perfusion; migration of shortened, cuboidal cells with blunt pseudopods and lamellipodia. This morphology predominated over sites of vascular congestion; and the formation of epithelial arches in which the epithelial cells did not make contact with the underlying basal lamina. Rapid repair by cell migration was observed only at sites in which a grossly intact basal lamina was present.

5 Functional Morphology of the Elasmobranch Nephron and Retention of Urea

Publisher Summary This chapter examines the functional morphology of the elasmobranch nephron and the retention of urea. The three organs that are used to maintain osmotic homeostasis in elasmobranch fish include the rectal gland, gill, and kidney. The kidneys are paired organs flanking the dorsal aorta in the abdomino-pelvic region. They are roughly semilunar in most skates, elongated in sharks, but always dorsoventrally flattened. In the spiny dogfish, segmental arteries of the dorsal aorta branch to give off renal arteries, each of which gives off two branches, one going caudad and other cephalad. The segregation of nephron segments that occurs in the bundle zone of marine species is not present in the sinus zone. Tubules from many different nephrons randomly intermingle in a large vascular sinus that appears to be continuous throughout this zone of the kidney. In marine elasmobranchs as in higher vertebrates, the juxtaglomerular apparatus lies at the vascular pole of the renal corpuscle and is composed of afferent arteriole with granular smooth muscle cells in its tunica media, efferent arteriole, macula densa of the distal tubule, and extraglomerular mesangium.

Granulated peripolar epithelial cells in the renal corpuscle of marine elasmobranch fish

SummaryGranulated epithelial cells at the vascular pole of the renal corpuscle, peripolar cells, have been found in the kidneys of five species of elasmobranchs, the little skate (Raja erinaced), the smooth dogfish shark (Mustelus canis), the Atlantic sharpnose shark (Rhizoprionodon terraenovae), the scalloped hammerhead shark (Sphryna lewini), and the cow-nosed ray (Rhinoptera bonasus). In a sixth elasmobranch, the spiny dogfish shark (Squalus acanthias), the peripolar cells could not be identified among numerous other granulated epithelial cells. The peripolar cells are located at the transition between the parietal epithelium of Bowmans capsule and the visceral epithelium (podocytes) of the glomerulus, thus forming a cuff-like arrangement surrounding the hilar vessels of the renal corpuscle. These cells may have granules and/or vacuoles. Electron microscopy shows that the granules are membrane-bounded, and contain either a homogeneous material or a paracrystalline structure with a repeating period of about 18 nm. The vacuoles are electron lucent or may contain remnants of a granule. These epithelial cells lie close to the granulated cells of the glomerular afferent arteriole. They correspond to the granular peripolar cells of the mammalian, avian and amphibian kidney. The present study is the first reported occurrence of peripolar cells in a marine organism or in either bony or cartilagenous fish.

Effects of misoprostol, cimetidine, and ethanol on rat gastric plasma volume and morphology

Prostaglandins have effects on the gastric vasculature and blood flow that may be related to the phenomenon of “cytoprotection.” We evaluated the effects of two potent antiulcer compounds—misoprostol, a prostaglandin E1 analog, and cimetidine, an H2-receptor antagonist—as well as a necrotizing agent, absolute ethanol (EtOH), on gastric plasma volume using the Evans blue-albumin binding technique. In addition, the effects of these two drugs on the morphology of the gastric mucosa were evaluated by light microscopy of epoxy resin sections. Fasted rats were given one of the following oral doses: (a) misoprostol, subantisecretory (100 μg/kg) or antisecretory (1 mg/kg); (b) cimetidine, subantisecretory (100 μg/kg, or 1 mg/kg) or antisecretory (50 mg/kg): or (c) EtOH only or EtOH following 30 min of pretreatment with one of the above doses of misoprostol or cimetidine. Animals were killed 30 or 90 min after a single dose or 30 min after two doses that were 60 min apart. The stomachs were then removed and processed for biochemical determination of Evans blue concentration. For morphology, rat stomachs were fixed and processed after exposure to each of the above concentrations of misoprostol and cimetidine. Results were as follows: (i) Misoprostol (but not cimetidine) caused a significant increase in stomach weight (subantisecretory dose = +15%, antisecretory dose = +21%) independent of changes in plasma volume. (ii) Misoprostol at both doses caused a transient (30-min) reduction in plasma volume (subantisecretory dose = −12%, antisecretory dose = −14%) that rebounded to slightly elevated levels within the following 60 min. (iii) Antisecretory doses of cimetidine caused an increase in plasma volume 30 min after a single dose (+ 37%) and at 90 min after two doses (+46%). (iv) EtOH only at 30 or 90 min produced elevated stomach weights (+25 and +28%, respectively) and plasma volumes (+300 and +250%, respectively), the latter of which was due in part to hemorrhagic lesions. (v) Cimetidine pretreatment (30 min) followed by EtOH did not significantly reduce plasma volume, stomach weight, or the gross hemorrhagic lesions compared to EtOH alone, but (vi) misoprostol pretreatment followed by EtOH significantly protected against hemorrhagic lesions and elevated tissue plasma volumes. Both drugs at all concentrations caused expansion of the mucosal interstitial space (lamina propria), but this edema was greater with misoprostol. Severe interfoveolar cell stretching and even epithelial breaks were found with misoprostol treatment. Cimetidine showed moderate mucosal edema and a “collapse” of interfoveolar epithelial cells so the intercellular spaces were reduced. We conclude that there is no correlation between changes in rat gastric plasma (blood) volume induced by either misoprostol or cimetidine and protection against an acute insult of EtOH. Misoprostol appears to protect the stomach in part by preventing microvascular stasis and subsequent hemorrhagic lesions during EtOH challenge independent of the amount or velocity of the blood flowing through the organ. However, misoprostol induced rapid tissue weight increases exclusive of changes in plasma volume, an effect that may be related secondarily to its protective action via the extensive mucosal edema it causes.