Gastric mucosal lesions after burn injury: relationship to H+ back-diffusion and the microcirculation

Rats were subjected to a 30% body surface area full-thickness burn. Two hours after injury, 93% of animals had gastric mucosal erosions. At 5 hours this increased to 100%, but at 24 and 72 hours, lesions were fewer and less severe. Histologic study suggested that lesions noted at 24 and 72 hours represented erosions formed earlier. No mucosal abnormalities were noted in control rats. A causal relationship between mucosal ischemia and the development of erosions is suggested by the presence of A-V shunts at 2 and 5 hours only. Significant increases in H+ back-diffusion and protein leakage into the gastric lumen at 2 and 5 hours also implicated changed mucosal permeability in the etiology of erosions. The return of H+ back-diffusion to control values at 24 and 72 hours, when lesions were still present, appears to contradict the theory that permeability changes are secondary to erosion formation.     

Treatment of experimental mouse bladder tumour by LPS-induced epithelial cell shedding

The purpose of the present study was to explore the therapeutic potential of serial administration of shedding-inducing endotoxin in a mouse tumour bladder model. The studies were conducted with two variants derived from the MBT-2 tumour namely, T5 and T50, the latter being far more aggressive than the former. It was found that T5 tumours responded to intravesical lipopolysaccharides (LPS) instillation by a considerable reduction in their pace of growth (P < 0.0001) when treatment was initiated 3 days after tumour implantation, but not when started after 7 days. The T50 variant did not respond to LPS when treated 3 days after implantation, but a considerable reduction in rate of growth occurred when treatment was started after 1-2 days. Shedding induced by intravesically instilled LPS was found to retard considerably the progression rate of experimental bladder tumour.     

Effect of intravesical capsaicin and vehicle on bladder integrity control and spinal cord injured rats

PURPOSE: To determine the acute effect of intravesical capsaicin on bladder mucosal integrity in normal and spinal cord injured (SCI) rats. MATERIALS AND METHODS: Intravesical reagents were instilled in 5 groups of age and weight matched female rats: 1) control + normal saline solution (NSS), 2) control + ethanol (EtOH), 3) control + capsaicin/EtOH, 4) SCI + NSS, 5) SCI + capsaicin/EtOH. Intravesical instillations were performed 4 weeks after a standard T10 SCI. Intravesical capsaicin (1 mM.) was dissolved in 30% EtOH/NSS. The animals (n = 3 each group) were sacrificed at 30 minutes, 24 hours, 72 hours, and 7 days after intravesical instillation. Whole bladders were harvested, fixed in 10% buffered formalin, and paraffin embedded. Tissue blocks were blind coded and sectioned (5 microns thickness) for histopathological analysis. All sections were initially stained with hematoxylin and eosin (H & E). Specific staining for mucin carbohydrate moieties included periodic acid-Schiff (PAS) and alcian blue. Also, immunohistochemical staining for GP51 (a urinary glycoprotein) was performed. RESULTS: Control and SCI rats exhibited similar bladder mucosal histology by H & E and mucin specific stains. Instillation of saline demonstrated no effect on bladder histology, whereas instillation of intravesical capsaicin induced a profound acute effect of thinning of the epithelium, submucosal edema, and diminished presence of GP51. EtOH produced similar pathological findings, but to a lesser degree than capsaicin. Intravesical capsaicin demonstrated a similar effect in both control and SCI animals. The peak effect was seen after 30 minutes and continued for 24 hours. Partial recovery was noted after 72 hours and complete recovery was evident by 1 week. CONCLUSIONS: The control and SCI rats demonstrated a histologically similar mucosa and glycosaminoglycan layer. The effect of saline instillation on the mucosa was negligible. Intravesical capsaicin dissolved in 30% ethanol/NSS had a profound effect on the bladder urothelium submucosa that was more pronounced than that seen with the ethanol vehicle alone in normal animals.     

Matrix metalloproteinases and TIMPs are associated with blood-brain barrier opening after reperfusion in rat brain

BACKGROUND and PURPOSE: Reperfusion disrupts cerebral capillaries, causing cerebral edema and hemorrhage. Middle cerebral artery occlusion (MCAO) induces the matrix-degrading metalloproteinases, but their role in capillary injury after reperfusion is unknown. Matrix metalloproteinases (MMPs) and tissue inhibitors to metalloproteinases (TIMPs) modulate capillary permeability. Therefore, we measured blood-brain barrier (BBB) permeability, brain water and electrolytes, MMPs, and TIMPs at multiple times after reperfusion. METHODS: Adult rats underwent MCAO for 2 hours by the suture method. Brain uptake of 14C-sucrose was measured from 3 hours to 14 days after reperfusion. Levels of MMPs and TIMPs were measured by zymography and reverse zymography, respectively, in contiguous tissues. Other rats had water and electrolytes measured at 3, 24, or 48 hours after reperfusion. Treatment with a synthetic MMP inhibitor, BB-1101, on BBB permeability and cerebral edema was studied. RESULTS: Brain sucrose uptake increased after 3 and 48 hours of reperfusion, with maximal opening at 48 hours and return to normal by 14 days. There was a correlation between the levels of gelatinase A at 3 hours and the sucrose uptake (P<0.05). Gelatinase A (MMP-2) was maximally increased at 5 days, and TIMP-2 was highest at 5 days. Gelatinase B and TIMP-1 were maximally elevated at 48 hours. The inhibitor of gelatinase B, TIMP-1, was also increased at 48 hours. Treatment with BB-1101 reduced BBB opening at 3 hours and brain edema at 24 hours, but neither was affected at 48 hours. CONCLUSIONS: The initial opening at 3 hours correlated with gelatinase A levels and was blocked by a synthetic MMP inhibitor. The delayed opening, which was associated with elevated levels of gelatinase B, failed to respond to the MMP inhibitor, suggesting different mechanisms of injury for the biphasic BBB injury.     

Increased plasma P-selectin induced by intravenous administration of endotoxin in rats

We developed a competitive enzyme immunoassay for detection of rat P-selectin and examined temporal changes of plasma P-selectin levels in an endotoxin-induced injury model in rats. Soluble P-selectin was detected in rat plasma after intravenous administration of lipopolysaccharide (LPS), and increased to a maximum level of five-fold over baseline after 24 hours. Plasma P-selectin was partially purified by gelfiltration and was identified as a 122 kDa band by Western blotting. Using the ELISA system we developed, monitoring of plasma P-selectin has become possible in the rat.     

Vulnerability to cerebral hypoxic-ischemic insult in neonatal but not in adult rats is in parallel with disruption of the blood-brain barrier

BACKGROUND AND PURPOSE: Vulnerability to cerebral hypoxic-ischemic (H-I) insult and its relation to disruption of the blood-brain barrier were investigated in postnatal rats. METHODS: Pups of postnatal day (P) 7, P14, and P21 underwent ligation of a unilateral carotid artery and were exposed to hypoxic conditions. For the detection of early-phase deterioration, brains were perfusion-fixed 24 hours after H-I insult and examined by argyrophil III method. For the detection of later infarction, animals were fixed at 72 hours after the H-I insult. RESULTS: In either case, tissue damage was detected in the striatum, parietal cortex, and hippocampus. The vulnerability of P7 and P21 rats was remarkable, as compared with P14 rats. Although the developmental status of the vasculature was not significantly different at each age, the permeability of IgG after H-I injury was prominent in P7 rats and to a lesser extent in P14 rats. In P21 rats, however, there was little IgG leakage even 24 hours after the insult. Dexamethasone pretreatment blocked the extravasation of IgG and reduced the damaged tissue in P7 and P14 rats but not in P21 rats. Percentages of reduction in infarcted areas by the dexamethasone became smaller in proportion to ages. CONCLUSIONS: The results suggest that in younger rats vulnerability to H-I insult was in parallel with permeability of the blood-brain barrier, whereas in adults in might be more dependent on cellular vulnerability.     

Leukemic pleural effusion in B-cell prolymphocytic leukemia

BACKGROUND: Proinflammatory mediators that include tumor necrosis factor-alpha (TNF-alpha) and macrophage inflammatory protein-2 (MIP-2) and anti-inflammatory mediators such as interleukin-10 (IL-10) modulate the immune response to endotoxemia. IL-10 downregulates the production of TNF-alpha and MIP-2. Acute lung injury may occur secondary to neutrophil chemotaxis mediated by chemokine MIP-2. We studied the temporal relationship of TNF-alpha, MIP-2, and IL-10 in rat endotoxemia and correlation of MIP-2 concentrations with acute lung injury. METHODS: Ten ventilated rats were randomized to receive an intravenous infusion of 2 mg/kg Escherichia coli lipopolysaccharide (n = 6) or saline placebo (n = 4). Blood pressure was continuously monitored and arterial blood was obtained for lactate, blood gas, TNF-alpha, IL-10, and MIP-2 measurements at baseline, 2, 4, and 5.5 hours after LPS or saline infusion. RESULTS: Endotoxemia resulted in hypotension, lactic acidemia, and increased alveolar-arterial oxygen gradient (A-a O2 gradient) compared with the placebo group. TNF-alpha, MIP-2, and IL-10 levels were increased 2 hours after endotoxemia. Subsequently, TNF-alpha levels declined while IL-10 and MIP-2 levels remained elevated. Control rats had no significant increase in cytokine production at any time point. MIP-2 concentrations correlated with A-a O2 gradient, an indicator of lung injury (r = 0.56, p < 0.001). CONCLUSIONS: MIP-2, possibly released by TNF-alpha stimulation of macrophages, is associated with acute lung injury possibly by inducing neutrophil chemotaxis. IL-10 may exert its counter-inflammatory response by inhibiting the release of TNF-alpha in endotoxemia.     

Photodynamic therapy on rat urinary bladder with intravesical instillation of 5-aminolevulinic acid: light diffusion and histological changes

PURPOSE: Photodynamic therapy (PDT) has the potential to treat extensive premalignant lesions and microinvasive tumors in the bladder, but its use has been hampered by the risk of detrusor muscle damage and prolonged skin photosensitivity. We have shown that the rat urothelium can be sensitized by selectively using a 10% solution of 5-aminolevulinic acid (ALA) at pH 5.5 administered intravesically. This paper evaluates the photodynamic effects on sensitized bladders. MATERIALS AND METHODS: The bladders ofs Wistar rats were instilled with ALA solutions of different concentrations at pH 5.5 and subsequently treated with laser light at 630 nm. Bladders were harvested 1 to 7 days after PDT for histological assessment. RESULTS: Under optimum conditions (10% intralipid diffusion medium, light dose 50J) uniform urothelial necrosis was seen after 1 to 2 days; it healed in 7 days without damage to the underlying muscle layer although some increase in collagen was seen in the lamina propria. Overtreatment or poor light distribution resulted in muscle necrosis and scarring. CONCLUSIONS: Selective urothelial necrosis is possible with PDT using intravesical ALA. There is now sufficient data for pilot clinical trials to start photodynamic therapy for management of superficial bladder cancer or carcinoma in situ.     

Evaluation of smooth muscle and collagen subtypes in normal newborns and those with bladder exstrophy

PURPOSE: Many patients who undergo bladder exstrophy closure as newborns, subsequent epispadias repair and later bladder neck reconstruction become completely continent yet complications can occur. After successful initial exstrophy closure and later epispadias repair some patients may fail to gain sufficient capacity for bladder neck reconstruction or satisfactory capacity and continence after bladder neck reconstruction. In an attempt to understand the pathogenesis of these failures we compared bladder biopsies from normal neonates and those with exstrophy. MATERIALS AND METHODS: Bladder biopsies obtained from the midline of the bladder wall just above the base of the trigone from 12 newborns with exstrophy were compared to bladder sections from 9 neonatal cadavers. All bladder specimens were stained with monoclonal antibodies against type I, III or IV collagen and a subset was further stained with Masson's trichrome to define the extracellular matrix. All specimens were then analyzed using a color digital image analysis system. RESULTS: At initial examination of the extracellular matrix there was an increase in the collagen-to-smooth muscle ratio from 0.38 in controls to 1.2 in newborns with exstrophy, comprising an increase in collagen and decrease in smooth muscle. The collagen component of the extracellular matrix was then further defined to quantitate the amount of each collagen type (I, III and IV) deposited. We then evaluated the ratio of collagen type-to-total collagen sampled. Compared to control bladders there was no statistical difference in the amount of type I or IV in the bladders of newborns with exstrophy at initial closure. However, there was a 3-fold increase in type III collagen (0.14 +/- 0.05 to 0.46 +/- 0.2%, p < 0.001) in the bladders of neonatal controls versus newborns with exstrophy. CONCLUSIONS: This alteration in collagen makeup may represent an earlier developmental stage of the exstrophy bladder at birth, which then remodels and changes after successful initial closure. Further studies are underway to examine the collagen composition of bladders at bladder neck reconstruction, failed closures and augmentation.     

Protection from renal ischemia-reperfusion injury by the 2-methylaminochroman U83836E

BACKGROUND: In a prior study the 21-aminosteroid (lazaroid) U74389F provided in vivo protection from oxidative stress when used as a preventive therapy in ischemia-reperfusion injury in the kidney. As the cell membrane is the principal site for lipoperoxidation, in the current study the very lipophilic 2-methylaminochroman U83836E, a recently developed lazaroid, was administered to rats at 3 mg/kg before renal ischemia-reperfusion. In addition to the biochemical parameters, the renal function and the histological appearance were carefully evaluated. METHODS: Glutathione, adenine nucleotides and lipid peroxidation products were determined in kidneys reperfused for 2 and 24 hours after 90 minutes of ischemia. Renal function was assessed by plasma creatinine, and renal injury by histological examination. RESULTS: Reperfusion-induced glutathione oxidation, expressed as an oxidized-to-total glutathione ratio, was significantly attenuated both after 2 and 24 hours of reperfusion by treatment with U83836E. Adenosine triphosphate (ATP) was still significantly depleted after 24 hours in the control group, while at the same time treated animals had already recovered to baseline values. Lipid peroxidation products were significantly lower in lazaroid-groups both after 2 and 24 hours of reperfusion. Renal function after 24 hours of reperfusion was notably better in the treated rats. Histological examination confirmed the protective action of the drug. After 24 hours the control group showed large areas of parenchymal hemorrhage and necrosis with dilated tubules and blood vessel thrombosis, while treated animals showed small necrotic areas with a background of mild interstitial inflammatory cells. CONCLUSIONS: Our results suggest that there is a protective effect of U83836E in ischemia-reperfusion injury, in that tissue damage due to oxidative stress is reduced, thus ameliorating renal function impairment.     

Induction of glutamine synthetase expression after major burn injury is tissue specific and temporally variable

BACKGROUND: Major burn injury results in a translocation of amino acids from peripheral tissues to the abdominal viscera. Glutamine is a major participant in this event. Thermal injury causes a depletion of plasma and muscle glutamine pools as well as activation of proteolysis and release of glutamine from skeletal muscle. De novo synthesis of glutamine is regulated by the expression of the enzyme glutamine synthetase (GS). We studied the tissue-specific regulation of GS expression after thermal injury. METHODS: Burn injury of rats was produced by scalding of 25 or 40% of skin surface. In normal rats, four organs, including lung, muscle, kidney, and liver were assayed for relative GS messenger RNA content by Northern blotting 8 and 24 hours after 40% area burn. The effect of adrenalectomy on GS mRNA induction in muscle was assessed 24 hours after 25% area burn injury. RESULTS: GS mRNA levels were increased 2.3-fold in lung at 8 hours and 7.3-fold in muscle at 24 hours after burn injury. No appreciable increase in GS mRNA level was observed in kidney or liver. Muscle GS mRNA levels were lower than sham-operated controls in both burned and unburned adrenalectomized rats. However, adrenalectomy did not attenuate relative GS mRNA induction in muscle at 24 hours after burn injury. CONCLUSIONS: Burn injury causes an induction in GS mRNA levels in a tissue-specific fashion. Adrenalectomy greatly reduced GS mRNA levels, but did not completely block the induction of GS express in muscle after burn injury. This finding suggests that glucocorticoid hormones together with a unknown factor of nonadrenal origin influence this metabolic response to burn injury.     

Bladder instillation and intraperitoneal injection of Escherichia coli lipopolysaccharide up-regulate cytokines and iNOS in rat urinary bladder

Systemic bacterial lipopolysaccharides (LPS) induce inflammatory responses characteristic of sepsis. Instillation of LPS into rat bladder produces a localized inflammatory response similar to that seen in urinary tract infections (UTIs). Four hours after intravesical instillation of LPS, neutrophils infiltrate into the bladder, and mRNA for inducible nitric oxide synthase (iNOS) and the cytokines, interleukin (IL)-6 and IL-10, is detected in rat bladder but not in the kidney. Induction of iNOS protein is inferred because urinary nitrate and cGMP levels are increased 4 hr after LPS intravesical instillation and remain elevated for at least 24 hr. When LPS is injected intraperitoneally, iNOS and IL-6 mRNA are induced both in the bladder and in the kidney. These data are consistent with the effects of intravesical instillation of LPS remaining localized, iNOS activity increases in both particulate and soluble bladder fractions when measured 4 hr after intravesical instillation of LPS. The magnitude of these increases in iNOS activity in the bladder is not as great as when LPS is injected intraperitoneally. Intravesical instillation of LPS induces no increase in lung or kidney NOS activity. The localized inflammatory response produced by intravesical instillation of LPS demonstrates the importance of LPS as a mediator of the host response in UTIs and supports the use of urinary measurements of nitrate and cGMP in humans as indicative of the localized induction of iNOS in UTIs.     

Transient alteration in intestinal permeability to technetium Tc99m diethylenetriaminopentaacetate during the prodromal stages of alimentary laminitis in ponies

OBJECTIVES: To determine whether mucosal permeability is altered during the prodromal stages of alimentary laminitis. ANIMALS: 15 healthy adult ponies. PROCEDURES: intestinal permeability was evaluated for control ponies (n = 5) and for ponies 4 to 12 (n = 5) and 20 to 28 (n = 5) hours after administration of carbohydrate overload. Mucosal permeability was determined by measuring the percentage of orally administered technetium Tc99m diethylenetriaminopentaacetate (99mTc-DTPA) excreted in urine during an 8-hour period, then measuring blood radioactivity at hourly intervals. Plasma endotoxin-like activity was measured by use of a chromogenic Limulus amebocyte assay. RESULTS: Urinary excretion of 99mTc-DTPA was 2.45% of administered dose for control ponies, and was 16.67% of administered dose 4 to 12 hours and 3.57% of administered dose 20 to 28 hours after administration of carbohydrate. CONCLUSIONS: A marked but transient increase in intestinal permeability was observed early in the prodromal stages of alimentary laminitis. CLINICAL RELEVANCE: Absorption of substances from the intestine may be an initiating event in alimentary laminitis.     

Esophagitis in Sprague-Dawley rats is mediated by free radicals

Free radical-mediated esophagitis was studied during duodenogastroesophageal reflux (mixed reflux) or acid reflux in rats. The influence of reflux on esophageal glutathione levels was also examined. Mixed reflux caused more gross mucosal injury than acid reflux. Gross mucosal injury occurred in the mid-esophagus. Total glutathione (GSH) in the esophageal mucosa of control rats was highest in the distal esophagus. The time course of esophageal GSH in rats treated by mixed reflux showed a significant decrease 4 hr after initiation of reflux, followed by a significant increase from the 12th hour on. Mucosal GSH was increased in both reflux groups after 24 hr but significantly more so in the mixed than in the acid reflux group. The free radical scavenger superoxide dismutase (SOD) prevented esophagitis and was associated with decreased GSH levels. GSH depletion by buthionine sulfoximine (BSO) prevented esophagitis and stimulated SOD production in the esophageal mucosa. It is concluded that gastroesophageal reflux is associated with oxidative stress in the esophageal mucosa. The lower GSH levels in the mid-esophagus may predispose to damage in this area. Duodenogastroesophageal reflux causes more damage than pure acid reflux. Oxidative stress leads to GSH depletion of the esophageal mucosa in the first few hours following damage but then stimulates GSH production. GSH depletion by BSO does not worsen esophagitis since it increases the esophageal SOD concentration.     

An essential role for free radicals and derived species in signal transduction

OBJECTIVE: To examine whether there is generation of oxygen free radicals (OFR) and lipid peroxidation of cell membrane after volume replacement for burn shock, and to study the relationship between OFR injury and enterogenous endotoxemia. METHODS: Forty-seven burn patients were involved in this study. Among them, 18 had delayed fluid resuscitation (DR) and the others had early fluid resuscitation (ER) within 6 hours postburn. Sixty-six gnotobiotic rats were used in a collaborating experiment as burn models. They were divided into 4 groups: sham injury (n = 6), early resuscitation (n = 24), late resuscitation (n = 24) and vitamins E and C treatment group (n = 12). All the rats, except those in the sham injury group, were inflicted with 40% total body surface area (TBSA) third-degree burns. OFR was determined in the blood of patients with electron spin resonance (ESR). S/W ratio and tau c values of patients' erythrocytes were measured with ESR spectrometer. Blood superoxide dismutase (SOD) and glutathione peroxidase (GSHPx) activities, malondialdehyde contents and plasma endotoxin levels were assayed. Rats were sacrificed at the 12th, 24th, 48th and 72nd hour after injury. Plasma endotoxin levels, mucosal SOD, GSHPx and malondialdehyde (MDA), as well as diamine oxidase activity of ileum were determined. Cultures of mesenteric lymph nodes (MLN), liver, spleen, heart, lung, kidney and blood were done. RESULTS: A significant increase in blood OFR contents and plasma MDA, and a significant decline in blood SOD and GSHPx were found after resuscitation in DR group as compared with those in ER group. Both strong to weak spectra component (S/W) ratio and tau c value were higher in DR group in contrast with those in ER group. Higher elevation in plasma endotoxin level in DR group was seen. In DR group, plasma MDA content was correlated with S/W ratio, tau c value and plasma endotoxin level. In rats, the level of mucosal MDA, plasma endotoxin and incidence of bacterial translocation (BT) were significantly higher. Mucosal SOD, GSHPx and diamine oxidase (DAO) activity were significantly lower in DR group as compared with those in ER group. In DR group, mucosal MDA content was negatively correlated with mucosal DAO activity, while the latter was negatively correlated with BT. After treatment with vitamins E and C, mucosal MDA content decreased, plasma endotoxin and BT significantly declined and mucosal DAO heightened. CONCLUSIONS: Tissue reperfusion might induce the production of OFR, resulting in lipid peroxidation injury, especially to intestinal mucosa, and resulting in disruption of mucosal barrier function followed by endotoxemia and BT.