Sean M. Oakes

ATTENUATION OF THE EFFECTS OF RAT HEMORRHAGIC SHOCK WITH A REPERFUSION INJURY-INHIBITING AGENT SPECIFIC TO MICE

Death after hemorrhagic shock (HS) may be caused by a generalized reperfusion injury, particularly noticeable in the gut. A period of tissue ischemia followed by reinstitution of perfusion produces severe inflammation that can be blocked in mice by preventing the binding of a pathogenic natural immunoglobulin M (IgM) of defined specificity to antigens in reperfused tissue by using a soluble peptide analogue of the IgM tissue target. We hypothesize that this agent can improve end points of rat HS: death, intestinal injury, and lung injury. Male Sprague-Dawley rats were anesthetized; 50% of calculated blood volume was removed for 120 min, shed blood, then returned; and animals were sacrificed at 72 h. One group of rats received i.v. analogue ([N2] 300 &mgr;g) with the return of shed blood. Small intestine and lung were evaluated by histological examination and immunohistochemistry. Lung edema was assessed by Evans blue extravasation and histological examination. I.v. N2 decreased experimental mortality from 62% to 12% (P < 0.05). Associated with this was diminution of gut injury score from 57.8% ± 5.5% to 19.5% ± 2.5% (P < 0.05), lung injury from 21.4 ± 1.5 to 14.8 ± 1.3 polymorphonuclear leucocytes per high-power field ×400 (P < 0.05), and Evans blue extravasation index from 0.61 ± 0.14 to 0.18 ± 0.06 (P < 0.05). As well, the deposition of IgM and C3 that is seen in intestinal villi from HS was not present in N2-treated rats. The N2 peptide agent that blocks reperfusion injury in mice prevents death from rat HS, as well as attenuates gut reperfusion injury and its remote target injuries. These data suggest that death from HS is caused by reperfusion injury, and that an agent derived from mice is effective in rats when given in real therapeutic time.

Inhibition of rat gut reperfusion injury with an agent developed for the mouse. Evidence that amplification of injury by innate immunity is conserved between two animal species

Murine reperfusion injury follows binding of specific IgM natural antibodies to neo-antigens exposed in ischemic tissue. Peptides that mimic the site of antibody binding in the injury prevent IgM binding when administered intravenously before reperfusion. To determine whether this pathogenic sequence is restricted to mice, we have tested the ability of the peptide to prevent reperfusion injury in a dissimilar species, the rat. Sprague-Dawley rats were subjected to 40 min of mesenteric ischemia followed by 180 min of reperfusion. The peptide mimic was administered intravenously prior to reperfusion. Gut injury was quantified using a scoring system based on the hematoxylin-and-eosin section. (125)I-labeled albumin was used to assess local (gut) and remote (lung) injury. The macroscopic appearance of bowel from peptide-treated animals was less edematous and hemorrhagic. Microscopic analysis showed a significantly reduced injury score in peptide-treated animals. Permeability data indicated a significant reduction in local and remote injury in peptide-treated animals. The data demonstrate attenuation of rat gut microvillus injury, of gut edema, and of remote injury following mesenteric ischemia-reperfusion due to administration of an intravenous peptide mimic of a murine ischemia neo-antigen, indicating a second species uses a similar ischemia neo-antigen and corresponding natural antibody specificity to amplify reperfusion injury to the point of necrosis. This mechanism of inflammation is potentially applicable to higher species.

Abstract 3815: Murine tumors demonstrate variable responses to ischemia-reperfusion injury

Introduction: Acute ischemia/reperfusion (I/R) of normal tissues causes an inflammatory reaction initiated by binding of auto-reactive IgM antibody to an ischemia-induced self-antigen. Whether this inflammatory cascade also affects cancers - for example, after acute I/R during surgical resection or in conditions of chronic ischemia - is unclear. We examined the effect of I/R injury in three murine tumor models: B16F10 melanoma, Lewis lung carcinoma (LLC), and small intestinal adenomas of the APC min/+ mouse. Methods: LLC and B16F10 cell lines were implanted into the hindlimbs of C57BL/6 mice. Ischemia was induced by external tourniquet ligation of the femoral vessels. Small intestinal tumors in APC min/+ mice were subjected to ischemia by vascular clip occlusion of the superior mesenteric artery. Control animals underwent hindlimb ischemia to the non-tumor bearing hindlimb or laparotomy alone. Following graded times of ischemia and reperfusion, tumors and surrounding normal tissue were harvested for histological examination. Immunohistochemistry was performed for IgM and activated caspase-3. For hindlimb injury, mice were recovered and followed for LLC and B16F10 tumor growth. Results: Ischemia-reperfusion causes injury of LLC and B16F10 tumors. In contrast, intestinal adenomas of APC min/+ mice showed no signs of injury following ischemia, despite induction of necrosis and apoptosis of adjacent normal intestinal epithelium. Tumor susceptibility to ischemic injury correlated with immuno-histochemical deposition of IgM antibody. Ischemic injury of B16F10 significantly reduced tumor growth (36% volume reduction compared to control injury, p Conclusions: The three murine tumor models examined are each affected differently by an acute I/R injury, in contrast to the conserved inflammatory cascade induced in normal tissues following ischemia. APC min/+ adenomas evade ischemia-induced IgM binding and injury. While both LLC and B16F10 are acutely injured, the long term effects on growth are divergent. Targeted manipulation of the host inflammatory response to ischemia-reperfusion may be a method to influence tumor progression and growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3815.