Development of a murine model of blunt hepatic trauma

Abstract

Despite the prevalence of blunt hepatic trauma in humans, there are few rodent models of blunt trauma that can be used to study the associated inflammatory responses. We present a mouse model of blunt hepatic trauma that was created by using a cortical contusion device. Male mice were anesthetized with ketamine-xylazine-buprenorphine and placed in left lateral recumbency. A position of 2 mm ventral to the posterior axillary line and 5 mm caudal to the costal margin on the right side was targeted for impact. An impact velocity of 6 m/s and a piston depth of 12 mm produced a consistent pattern of hepatic injury with low mortality. All mice that recovered from anesthesia survived without complication for the length of the study. Mice were euthanized at various time points (n = 5 per group) until 7 d after injury for gross examination and collection of blood and peritoneal lavage fluids. Some mice were reanesthetized for serial monitoring of hepatic lesions via MRI. At 2 h after trauma, mice consistently displayed laceration, hematoma, and discoloration of the right lateral and caudate liver lobes, with intraabdominal hemorrhage but no other gross injuries. Blood and peritoneal lavage fluid were collected from all mice for cytokine analysis. At 2 h after trauma, there were significant increases in plasma IL10 as well as peritoneal lavage fluid IL6 and CXCL1/KC; however, these levels decreased within 24 h. At 7 d after trauma, the mice had regained body weight, and the hepatic lesions, which initially had increased in size during the first 48 h, had returned to their original size. In summary, this technique produced a reliable, low mortality, murine model that recreates features of blunt abdominal liver injury in human subjects with similar acute inflammatory response.

Figure 1.

Techniques for delivery of impact to create liver trauma. (A) The cortical contusion device, with piston lowered to within 12 mm of the body surface of the mouse. The mouse was secured, with the right forelimb extended forward, in a modified syringe case to allow exact positioning in left lateral recumbency. (B) The correct landmarks for impact were designated by a line extended from the caudal edge of the limb and axillary skin fold (dotted white line) and a second line denoted by palpation of the costochondral arch. A point 2 mm ventral to the posterior axillary line and 5 mm caudal to the ribs was the point of impact on the right side.

Figure 2.

Weight changes for mice after blunt hepatic trauma. After induction of anesthesia, mice were positioned for precise delivery of a blunt abdominal force from a specialized cortical contusion device. There was significant (*, P < 0.05) loss of body weight, although less than 10%, over the first 24 h which then demonstrated a significant rebound. Data are expressed as mean ± SEM (n = 5 per group).

Figure 3.

Plasma AST and ALT levels after blunt hepatic trauma. Mice were anesthetized for delivery of a blunt abdominal force to the right side. Within 2 h of injury, (A) ALT and (B) AST levels in plasma both were significantly (*, P < 0.05) elevated as compared with those in noninjured control animals. ALT levels decreased to those comparable to those of the control animals within 24 h, whereas AST levels required 48 to be significantly (P < 0.05) reduced. Data are expressed as mean ± SEM (n = 5 per group).

Figure 4.

Characterization of hepatic surface lesions after blunt trauma. Anesthetized mice received blunt abdominal force delivered to the right side and were euthanized at various time points thereafter. (A) The subsequent lesions were confined to the right lateral (RL), right medial (RM), and caudate lobes (CL), with no apparent damage to the right kidney (RK) or other organs. (B) The lesions included hematomas (large black arrow), discolorations (small black arrows), and lacerations (white arrow). (C) The surface areas of discolored lesions were measured by using a caliper. The affected surface area appeared to increase over the first 24 to 48 h after impact and significantly (*, P < 0.05 as compared with the value at the 2-h time point) decreased by 7 d after trauma (†, P < 0.05 as compared with the value for the 48-h group). Data are expressed as mean ± SEM (n = 5 per group).

Figure 5.

Histologic description of hepatic lesions. The liver was removed from mice at various time points after blunt abdominal trauma. (A) At 2 h after blunt-force trauma, the liver parenchyma has well-defined areas in which the normal architecture and cell structure appear necrotic, with little evidence of a local inflammatory reaction. (B) Within 24 h, the areas of necrosis have been infiltrated by polymorphonuclear cells. (D) After 7 d, the areas of necrosis are well-demarcated and surrounded by distinct bands of inflammatory cells, many of which are mononuclear. Photomicrographs are representative of at least 3 animals per group; magnification, 40×.

Figure 6.

MRI of hepatic trauma. Images were obtained from an anesthetized mouse at various time points after hepatic trauma from a cortical contusion device. (A) T1-weighted gradient-echo image of the right lateral and medial liver lobes at 2 h after injury, showing the hypointense lesion (arrow). (B) The same lesion shown as a T2-weighted fast spin-echo axial image 2 h after trauma. (C) T2-weighted image of the hyperintense lesion (arrow) at 48 h after trauma and (D) 7 d after injury. These images are representative of those from 3 animals per time point.

Figure 7.

Peripheral WBC counts after blunt trauma. Anesthetized mice underwent blunt abdominal force and were euthanized at various time points thereafter; CBC counts were performed on retroorbital blood samples taken immediately before euthanasia. (A) Total WBC, (B) neutrophil, (C) lymphocyte, and (D) monocyte counts after trauma were not significantly different from those before surgery (control values). (E) Eosinophil counts decreased (*, P < 0.05 compared with the value for the control group) after injury and remained low over the course of the study. Data are expressed as mean ± SEM (n = 5 per group).

Figure 8.

Peritoneal WBC counts after blunt trauma. At various time points after blunt force trauma, anesthetized mice were euthanized, and peritoneal lavage was performed for cell counts and differentials. (A) Macrophages. (B) Neutrophils. (C) Lymphocytes. (D) Eosinophil. Data are expressed as mean ± SEM (n = 5 per group); *, P < 0.05 between groups.

Figure 9.

Plasma cytokine levels after blunt trauma. Plasma was obtained from anesthetized mice at various time points after blunt injury to the liver, and ELISA were performed for detection of the proinflammatory cytokines (A) IL6, (B) IL1β, and (C) CXC1/KC and (D) the antiinflammatory cytokine IL10. Data are expressed as mean ± SEM (n = 5 per group); *, P < 0.05 compared with value for control group.

Figure 10.

Peritoneal cytokine levels after blunt trauma. Peritoneal lavage fluid was obtained from anesthetized mice at various time points after blunt injury to the liver, and ELISA were performed for detection of the proinflammatory cytokines (A) IL6, (B) IL1β, and (C) CXC1/KC and (D) the antiinflammatory cytokine IL10. Data are expressed as mean ± SEM (n = 5 per group); *, P < 0.05 between groups.