D. Neil Granger

Role of oxygen radicals in ischemia-induced lesions in the cat stomach

Ischemia in a stomach that contains acid may produce severe gastric mucosal injury. The extent to which oxygen-derived free radicals are involved in the pathogenesis of this injury was investigated in the present study. Local gastric ischemia was achieved by reducing celiac artery pressure to 30 mmHg for 1 h. Ischemic injury was assessed by recording the loss of 125I-albumin and 51Cr-red cells across the gastric mucosa. Cats were treated with a xanthine oxidase inhibitor (allopurinol), a superoxide radical scavenging enzyme (superoxide dismutase), and a scavenger of hydroxyl radicals (dimethyl sulfoxide). The damage associated with ischemia only occurred during reperfusion of the stomach and was worst in the antrum. The level of xanthine oxidase in the antrum was twice that of the corpus. Treatment with allopurinol, superoxide dismutase, and dimethyl sulfoxide reduced 51Cr-red cell loss to 15%, 25%, and 21% of control (untreated) animals, respectively. The data indicate that oxygen-derived free radicals play a role in ischemic injury to the stomach and that the hydroxyl radical, a secondary radical produced from the superoxide anion, appears to be the major oxygen radical contributing to ischemic damage.

Xanthine oxidase inhibitors attenuate ischemia-induced vascular permeability changes in the cat intestine

Previous reports indicate that allopurinol, a xanthine oxidase inhibitor, largely prevents the injury produced by reperfusion of ischemic tissues. In order to further assess the role of xanthine oxidase in ischemia-reperfusion injury, we examined the influence of another inhibitor of the enzyme (pterin aldehyde) on the increased vascular permeability produced by intestinal ischemia. Vascular permeability estimates in autoperfused segments of cat ileum were derived from the relationship between lymph-to-plasma protein concentration ratio and lymph flow. One hour of intestinal ischemia increased vascular permeability to 0.43 +/- 0.02 from a control (nonischemic) value of 0.08 +/- 0.005. In ischemic ileal segments pretreated with purified pterin aldehyde, vascular permeability increased to only 0.15 +/- 0.02. Pretreatment with commercially prepared folic acid, which is contaminated with pterin aldehyde, also attenuated the ischemia-induced increase in vascular permeability (0.16 +/- 0.04). These findings support the hypothesis that xanthine oxidase is a major source of oxygen-free radicals produced during reperfusion of the ischemic small bowel.

Gastric mucosal injury in the rat. Role of iron and xanthine oxidase

Recent studies have implicated oxygen free radicals in ischemia-reperfusion injury to the gastric mucosa. The aims of the present study were to test the hypothesis that the enzyme xanthine oxidase is the source of the oxygen radicals in the ischemic stomach and determine the importance of the iron-catalyzed Haber-Weiss reaction in generating the cytotoxic oxygen radicals. Gastric mucosal clearance of 51Cr-labeled red blood cells was measured during a 30-min control period, a 30-min ischemic period (hemorrhage to 25 mmHg arterial pressure), and a 60-80-min reperfusion period (reinfusion of shed blood). In untreated (control) rats, a dramatic rise (100-fold) in the leakage of 51Cr-labeled red blood cells into the gastric lumen was observed only during the reperfusion period. After the reperfusion period, gastric mucosal damage was further assessed using gross lesion area and histology. Rats were placed on a sodium tungstate diet (to inactivate xanthine oxidase), or treated with either deferoxamine (an iron chelating agent) or superoxide dismutase (a superoxide scavenger). All three interventions substantially reduced 51Cr-labeled red blood cell clearance and gross lesion area relative to untreated rats. However, tissue injury assessed histologically was similar in both treated and untreated animals. The results of this study support the hypothesis that oxygen free radicals mediate the hemorrhagic shock-induced extravasation of red blood cells. The data also indicate that xanthine oxidase is the source of the oxy-radicals and that the iron-catalyzed Haber-Weiss reaction is largely responsible for hydroxyl radical generation in this model.

Hypothermia prevents increased capillary permeability following ischemia-reperfusion injury☆

UNLABELLED Severely injured trauma victims are frequently hypothermic. It is unclear, however, whether hypothermia itself is a detrimental or protective physiologic response to injury. One of the major consequences of fluid resuscitation following ischemic injury is edema formation, characterized by ischemia-reperfusion injury models. The purpose of this study was to examine the effect of regional hypothermia on a feline intestinal model of ischemia-reperfusion injury. An autoperfused segment of cat ileum was isolated and arterial, venous, and lymphatic vessels were cannulated. Lymph flow (Q1), lymph (C1), and plasma (Cp) protein concentrations and segmental blood flow (Qb) were measured. Permeability changes were characterized by the minimal C1/Cp ratio obtained by elevating venous outflow pressure. Animals were divided into the following groups: Group I: 1 hr of intestinal ischemia (30 mm Hg) with autoreperfusion; Group II: 1 hr of intestinal hypothermia (28 degrees C) with subsequent rewarming; Group III: 1 hr of combined ischemia and hypothermia. Group III animals were either kept hypothermic (IIIA) or rewarmed (IIIB) during autoreperfusion. Minimal C1/Cp ratios (mean +/- SEM) were as follows: CONTROL 0.15 +/- 0.02; Group I*: 0.32 +/- 0.03; Group II: 0.15 +/- 0.01; Group IIIA: 0.18 +/- 0.02; Group IIIB*: 0.42 +/- 0.02; (* = P less than 0.01 vs control). Reperfusion flow rates were no different between Group IIIA and Group IIIB animals. Ischemia-reperfusion, but not hypothermia alone, caused a marked increase in intestinal capillary permeability. Permeability increased after combined ischemia and hypothermia only if reperfusion was accompanied by rewarming. Hypothermic reperfusion protected against the increased permeability following ischemia.

Permselectivity of the peritoneal membrane

To investigate the osmotic barrier characteristics of the peritoneal membrane during conditions similar to peritoneal dialysis in man, yet transperitoneal fluid movement was measured in 20 cats following intraabdominal placement of isotonic saline and hypertonic solutions of NaCl, glucose, raffinose, and inulin. Also, isooncotic solutions of hemoglobin and albumin and two sulfated high-molecular-weight dextrans were investigated. Transperitoneal fluid movement was measured by a volume recovery method. Oncotic pressures of test solutions and plasma were measured by osmometry. Peritoneal osmotic conductances were calculated from the rate of transperitoneal water movement and the difference in osmotic pressures between the test solution and isotonic saline. The average glucose osmotic conductance per unit body surface are was found to be 2.3 +/- 0.18 x 10(-3) ml . min-1 . mm Hg-1 . m-2, in good agreement with previous reports, and the glucose osmotic reflection coefficient (sigma) was estimated to be 0.02. All the osmotic conductances measured could be fitted to a peritoneal equivalent pore radius of approximately 6 nm according to current hydrodynamic theories. The peritoneal membrane filtration coefficient was estimated to be 0.12 ml . min-1 . mm Hg-1 . m-2, of which 0.5-1% was found to be due to transcellular water flow. In conclusion the results of this study indicate that the peritoneum is a highly selective membrane with restrictive properties comparable to those reported for continuous capillary beds.

Esophageal Blood Flow in the Cat: Normal Distribution and Effects of Acid Perfusion

The radioactive microsphere technique was used to estimate blood flow to different regions of the esophagus and to adjacent regions of the stomach before and after perfusion of the esophagus with hydrochloric acid (pH 1.5) for 5 min. Under resting conditions total blood flow, as well as blood flow to the mucosal-submucosal layer and the muscular layer, to both sphincters was significantly higher than to the esophageal body. Blood flow to the adjacent regions of the stomach was significantly higher than esophageal blood flow. Acid perfusion resulted in a large increase in total blood flow in both sphincters and the lower esophageal body. Gastric blood flow was not altered by acid perfusion. The esophageal hyperemia resulted primarily from an increase in blood flow to the muscular layer; mucosal-submucosal blood flow was increased only in the lower esophageal sphincter. The present study indicates that short periods (5 min) of gastroesophageal reflux may increase esophageal blood flow.

Villous motility: Relationship to lymph flow and blood flow in the dog jejunum

Villous contraction frequency, lymph flow, blood flow, and arteriovenous oxygen difference were measured in dog jejunum. Venous pressure elevation and plasma dilution were used to increase capillary fluid filtration. Both perturbations produced concomitant increases in villous contraction frequency and lymph flow. A highly significant correlation (r = 0.83, p less than 0.001) was obtained between villous contraction frequency and lymph flow. This finding, coupled with the observation that stimulation of net fluid absorption increases villous contraction frequency, suggests that villous motility is increased by a myogenic response elicited by increments in interstitial fluid pressure. In another series of experiments local arterial pressure was reduced in 20-mmHg steps from 120 to 20 mmHg. Although blood flow fell proportionately to arterial pressure, villous contraction frequency and oxygen uptake were maintained at a normal level when arterial pressure was between 120 and 60 mmHg. Villous motility and oxygen consumption fell progressively as arterial pressure was reduced below 60 mmHg. This observation indicates that ischemia does not alter villous contraction frequency unless blood flow is reduced below the level necessary to maintain normal tissue oxygenation.

Charge selectivity of rat intestinal capillaries: Influence of polycations

The role of fixed anionic sites on the intestinal capillary wall in transvascular protein exchange was assessed by neutralizing the negative charges with polycations. The studies were performed in anesthetized rats with an intestinal lymph cannula. Intestinal lymph flow and lymph and plasma total protein concentrations were measured at regular intervals before and after intravenous infusion of either protamine sulfate, poly-L-lysine, or poly-ethyleneimine. Protamine sulfate infusion produced an eightfold increase in lymph flow and a fivefold increase in lymph protein clearance. Lymph flow increased 4.6-fold and lymph protein clearance increased 3.6 times over control in rats receiving the poly-L-lysine infusion. Polyethyleneimine infusion produced results comparable in magnitude to protamine sulfate; however, the animals were unable to tolerate this agent. The enhanced transcapillary protein fluxes produced by the polycation infusions suggest that fixed anionic sites normally impede the egress of proteins from the intestinal vasculature.

Importance of Collateral Circulation in the Vascularly Occluded Feline Intestine

The aim of this study was to assess the extent of collateral blood flow provided by the celiac and inferior mesenteric arteries to the intestines during total occlusion of the superior mesenteric artery (SMA). In anesthetized cats, blood flow to the pancreas, duodenum, jejunum, ileum, and colon was measured with radioactive micropheres (15 microns in diameter) before and during occlusion of the SMA. Superior mesenteric artery occlusion significantly decreased (by 63%) blood flow to the head of the pancreas. Flow to the neck and tail of the pancreas was not altered. Blood flow to the proximal and distal duodenum was significantly reduced by 35% and 61%, respectively. Along the entire jejunum and ileum, SMA occlusion markedly decreased blood flow by an average of 71%. In the proximal colon, blood flow decreased by 63%, whereas flow to the middle and distal colon was not affected by SMA occlusion. Reduction in total wall blood flow to the small and large intestines was largely due to a marked reduction in mucosa/submucosa blood flow; muscularis/serosa flow was not affected. The results of this study suggest that total occlusion of the SMA does not compromise blood flow to the neck and tail of the pancreas and middle and distal colon (tissues that are normally perfused with blood from either the celiac or inferior mesenteric arteries). Perfusion through collaterals maintains flow to the head of the pancreas and gut (from duodenum to proximal colon) to within 30%-65% of control (preocclusion) flow. An important new observation of this study is that collateral blood vessels are much more effective in preventing ischemia in the muscularis/serosa than in the mucosa/submucosa after SMA occlusion.

Role of oxygen radicals in ischemic bowel disorders

Major consequences of ischemic bowel disorders are: enhanced transcapillary filtration, interstitial edema, and sequestration of fluid in the lumen of the bowel. Although several endogenous substances (histamine, prostaglandine, etc.) are released from the small bowel, specific inhibitors do not prevent increased intestinal vascular premeability produced by ischemia reperfusion. Cytotoxic oxygen metabolites — so-called oxygen free radicals — are generated in excess after ischemia during the early reperfusion period and endogenous defense mechanisms are overwhelmed. The highly reactive oxygen radicals damage proteins, lipids, carbohydrates, and nucleotides. Experimental studies showed that pretreatment with oxygen free radical scavengers (super-oxide dismutase, catalase, dimethylsulfoxide) provided significant protection againts ischemic injuries to the small bowel. Further studies gave evidence that the enzyme xanthine oxidase, which reacts with hypoxanthine, constitutes the primary source of oxygen radical production after intestinal ischemia. Xanthine oxidase inhibitors (allopurinol, pterin aldehyde) are powerful substances attenuating the ischemia-induced increase in intestinal vascular permeability. Morphometric studies on biopsies obtained after reperfusion of the intestine indicated likewise that pretreatment with superoxide dismutase, allopurinol, etc. largely prevented the typical intestinal epithelial necrosis induced by ischemia reperfusion. The experimental results are promising for further clinical research. It is conceivable that drugs such as allopurinol or superoxide dismutase may become useful as a treatment of ischemic gastrointestinal diseases, such as necrotizing enterocolitis, volvulus, and others.