A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.     

Murine Bioluminescent Hepatic Tumour Model

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. Download video file.^{(57M, mp4)}     

Development of a Murine Model of Blunt Hepatic Trauma

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.Abbreviation: CXCL1/KC, keratinocyte-derived chemokineTrauma is the most frequent cause of mortality worldwide,¹¹ and in cases of blunt abdominal trauma, the liver is the most frequently injured organ.⁴ In humans, traumatic injuries to the liver are graded (I through VI) according to the American Association for the Surgery of Trauma Liver Injury Scale, which is based on the severity of lesions, including hematomas, lacerations and vascular disruption.¹⁰ Parenchymal injuries (grades I through III) are more common than are major vascular injuries (grades V and VI), correlating with the greater hemodynamic stability and lower early mortality rates of parenchymal damage.⁴ Although early mortality rates may be low in low-grade injuries, the overall mortality rates for abdominal traumas involving liver are greater than that of abdominal trauma without liver damage. Late mortality after liver injury is associated with immunologic dysfunction, leading to systemic inflammatory response syndrome, sepsis, and multiple-organ failure.⁶To study the complex immune responses surrounding blunt hepatic trauma, an appropriate animal model is imperative, but few animal models of liver trauma have been described. Swine traditionally have been the preferred model because of similarity of the liver anatomy and lesions to those of human cases.² Nonpenetrating models have been developed in swine and involve impact by crossbow or other blunted projectiles.²⁰ The first rodent model of trauma was developed by positioning anesthetized rats under a column containing a flat weight.³ In that model, the severity of injury could be adjusted by the height of the column. This model was distinctive in its use of the least sentient species to date. The large animal models and even the rat model would readily support studies of treatment modalities and the measurement of hemodynamic parameters. For extensively characterizing the immunopathology associated with liver trauma, a murine model could offer distinct advantages, including the ready availability of transgenic mice and the extensive array of reagents for immunologic studies.The purpose of the current study was to develop a reliable and reproducible, closed abdominal, murine model of blunt hepatic trauma that is suitable for studies of posttraumatic immune dysfunction and related complications. Our first aim was to develop a low-mortality model that demonstrated gross and microscopic hepatic lesions similar to those seen in humans. The second study aim was to define selected systemic and local immune responses, including immune cell counts and cytokine levels. In addition, we examined the potential use of a noninvasive imaging technique (MRI) for the sequential evaluation of hepatic lesions.     

Temporal Expression of Chemokines Dictates the Hepatic Inflammatory Infiltrate in a Murine Model of Schistosomiasis

Schistosomiasis continues to be an important cause of parasitic morbidity and mortality world-wide. Determining the molecular mechanisms regulating the development of granulomas and fibrosis will be essential for understanding how schistosome antigens interact with the host environment. We report here the first whole genome microarray analysis of the murine liver during the progression of Schistosoma japonicum egg-induced granuloma formation and hepatic fibrosis. Our results reveal a distinct temporal relationship between the expression of chemokine subsets and the recruitment of cells to the infected liver. Genes up-regulated earlier in the response included T- and B-cell chemoattractants, reflecting the early recruitment of these cells illustrated by flow cytometry. The later phases of the response corresponded with peak recruitment of eosinophils, neutrophils, macrophages and myofibroblasts/hepatic stellate cells (HSCs) and the expression of chemokines with activity for these cells including CCL11 (eotaxin 1), members of the Monocyte-chemoattractant protein family (CCL7, CCL8, CCL12) and the Hepatic Stellate Cell/Fibrocyte chemoattractant CXCL1. Peak expression of macrophage chemoattractants (CCL6, CXCL14) and markers of alternatively activated macrophages (e.g. Retnla) during this later phase provides further evidence of a role for these cells in schistosome-induced pathology. Additionally, we demonstrate that CCL7 immunolocalises to the fibrotic zone of granulomas. Furthermore, striking up-regulation of neutrophil markers and the localisation of neutrophils and the neutrophil chemokine S100A8 to fibrotic areas suggest the involvement of neutrophils in S. japonicum-induced hepatic fibrosis. These results further our understanding of the immunopathogenic and, especially, chemokine signalling pathways that regulate the development of S. japonicum-induced granulomas and fibrosis and may provide correlative insight into the pathogenesis of other chronic inflammatory diseases of the liver where fibrosis is a common feature.     

A Murine Model of Hepatic Veno-occlusive Disease Induced by Allogeneic Hematopoietic Stem Cell Transplantation

Hepatic veno-occlusive disease (HVOD) is a life-threatening complication of bone marrow stem cell transplantation. The understanding of this clinical condition is hampered by the lack of suitable animal models. Here, we present a murine (BALB/c-based) model of HVOD induced by allogeneic hematopoietic stem cell transplantation (allo-HSCT). The chimerism rate of bone marrow was measured on days 5 and 10, while the chimerism rate of peripheral blood was measured on day 15 after allo-HSCT. Percentages of peripheral reticulocytes and serum levels of bilirubin and alanine aminotransferase (as liver function tests) were measured on days 5, 10, 15, 20, and 30. Livers were obtained on days 5, 10, 15, 20, and 30, and fixed in formaldehyde or glutaric dialdehyde. Liver slices were processed using the hematoxylin–eosin, Masson’s trichrome, or immunohistochemistry staining, and examined by light or transmission electron microscopy. Sinusoidal damages were the earliest pathological changes occurring in the allo-HSCT-induced HVOD, followed by coagulative necrosis of liver cells. The liver cell necrosis was later attenuated and sinusoidal endothelial cell morphology improved. However, on day 30, the edema and necrosis of liver cells became aggravated again. Furthermore, sinusoidal lining cell regeneration and partly attenuated liver cell necrosis were followed by the moderate to severe central vein fibrosis. In conclusion, we have successfully established a murine model of HSCT-HVOD. This model develops moderate to severe HVOD which cannot heal without intervention.     

Lactoferrin Dampens High-Fructose Corn Syrup-Induced Hepatic Manifestations of the Metabolic Syndrome in a Murine Model

Hepatic manifestations of the metabolic syndrome are related obesity, type 2 diabetes/insulin resistance and non-alcoholic fatty liver disease. Here we investigated how the anti-inflammatory properties of lactoferrin can protect against the onset of hepatic manifestations of the metabolic syndrome by using a murine model administered with high-fructose corn syrup. Our results show that a high-fructose diet stimulates intestinal bacterial overgrowth and increases intestinal permeability, leading to the introduction of endotoxin into blood circulation and liver. Immunohistochemical staining of Toll-like receptor-4 and thymic stromal lymphopoietin indicated that lactoferrin can modulate lipopolysaccharide-mediated inflammatory cascade. The important regulatory roles are played by adipokines including interleukin-1β, interleukin-6, tumor necrosis factor-α, monocyte chemotactic protein-1, and adiponectin, ultimately reducing hepatitis and decreasing serum alanine aminotransferase release. These beneficial effects of lactoferrin related to the downregulation of the lipopolysaccharide-induced inflammatory cascade in the liver. Furthermore, lactoferrin reduced serum and hepatic triglycerides to prevent lipid accumulation in the liver, and reduced lipid peroxidation, resulting in 4-hydroxynonenal accumulation. Lactoferrin reduced oral glucose tolerance test and homeostasis model assessment-insulin resistance. Lactoferrin administration thus significantly lowered liver weight, resulting from a decrease in the triglyceride and cholesterol synthesis that activates hepatic steatosis. Taken together, these results suggest that lactoferrin protected against high-fructose corn syrup induced hepatic manifestations of the metabolic syndrome.     

Isoflurane and Sevoflurane Induce Severe Hepatic Insulin Resistance in a Canine Model

IntroductionAnesthesia induces insulin resistance, which may contribute to elevated blood glucose and adverse post-operative outcomes in critically ill patients, and impair glycemic control in surgical patients with diabetes. However, little is known about the mechanisms by which anesthesia impairs insulin sensitivity. Here we investigate the effects of anesthesia on insulin sensitivity in metabolic tissues.MethodsHyperinsulinemic-euglycemic clamps were performed in 32 lean (control diet; n = 16 conscious versus n = 16 anesthetized) and 24 fat-fed (6 weeks fat-feeding; n = 16 conscious versus n = 8 anesthetized) adult male mongrel dogs in conjunction with tracer methodology to differentiate hepatic versus peripheral insulin sensitivity. Propofol was administered as an intravenous bolus (3mg/kg) to initiate anesthesia, which was then maintained with inhaled sevoflurane or isoflurane (2–3%) for the duration of the procedure.ResultsAnesthesia reduced peripheral insulin sensitivity by approximately 50% in both lean and fat-fed animals as compared to conscious animals, and insulin action at the liver was almost completely suppressed during anesthesia such that hepatic insulin sensitivity was decreased by 75.5% and; 116.2% in lean and fat-fed groups, respectively.ConclusionInhaled anesthesia induces severe hepatic insulin resistance in a canine model. Countermeasures that preserve hepatic insulin sensitivity may represent a therapeutic target that could improve surgical outcomes in both diabetic and healthy patients.     

A Possible Murine Model for Investigation of Pathogenesis of Sudden Infant Death Syndrome

Several studies have indicated a possible causative role of toxigenic bacteria in sudden infant death syndrome (SIDS). This study examined the effect of toxigenic E. coli on pregnant and infant mice to determine if these animals could be used as a model for SIDS pathogenesis. Strains of E. coli from the intestinal contents of infants who have died of SIDS or other causes and from the faeces of healthy infants were collected over a broad time scale. The isolates were tested for their ability to produce then known toxins of E. coli and were serotyped (O and H antigens). Certain serotypes (e.g. O1:H- and O25:H1) emerged significantly more frequently from cases of SIDS than from healthy infants and isolates of these types were generally toxigenic in Vero-cell cultures but whose verotoxicity was not related to classical Shiga or other known toxins. This mouse model was developed to test the effects of these toxigenic and also non-toxigenic strains. Four apparently healthy pups aged between 17 and 21 days died unobserved overnight but no pups of the 54 control mice died suddenly (P = 0.0247, Fisher’s exact test). These were considered to represent sudden unexpected deaths. Pathological effects compatible with those in SIDS were observed in mouse pups exposed to toxigenic strains indicating this model may be suitable for further study into the pathogenesis of unexpected deaths in infancy. Providing an animal model of SIDS would promote a much better avenue for studying the pathogenesis of this enigmatic condition.     

IMD-0354 Targets Breast Cancer Stem Cells: A Novel Approach for an Adjuvant to Chemotherapy to Prevent Multidrug Resistance in a Murine Model

Although early detection of breast cancer improved in recent years, prognosis of patients with late stage breast cancer remains poor, mostly due to development of multidrug resistance (MDR) followed by tumor recurrence. Cancer stem cells (CSCs), with higher drug efflux capability and other stem cell-like properties, are concentrated in a side population (SP) of cells, which were proposed to be responsible for MDR and tumor repopulation that cause patients to succumb to breast cancer. Therefore, targeting of CSCs as an adjuvant to chemotherapy should be able to provide a more effective treatment of this disease. Here, we used IMD-0354, an inhibitor of NF-κB, identified for targeting CSCs, in a combination therapy with doxorubicin encapsulated in targeted nanoparticles. IMD-0354 did target CSCs, evidenced by a decrease in the SP, demonstrated by the inhibition of the following: dye/drug efflux, reduction in ABC transporters as well as in colony formation in soft agar and low attachment plates. Decrease of stem-like gene expression of Oct4, Nanog and Sox2, and apoptosis resistance related to the Survivin gene also was observed after treatment with this compound. In addition, IMD-0354 targeted non-CSCs as indicated by reducing viability and increasing apoptosis. Targeted drug delivery, achieved with a legumain inhibitor, proved to enhance drug delivery under hypoxia, a hallmark of the tumor microenvironment, but not under normoxia. Together, this allowed a safe, non-toxic delivery of both anticancer agents to the tumor microenvironment of mice bearing syngeneic metastatic breast cancer. Targeting both bulk tumor cells with a chemotherapeutic agent and CSCs with IMD-0354 should be able to reduce MDR. This could eventually result in decreasing tumor recurrences and/or improve the outcome of metastatic disease.     

In Vivo MRI Assessment of Hepatic and Splenic Disease in a Murine Model of Schistosmiasis

BackgroundSchistosomiasis (or bilharzia), a major parasitic disease, affects more than 260 million people worldwide. In chronic cases of intestinal schistosomiasis caused by trematodes of the Schistosoma genus, hepatic fibrosis develops as a host immune response to the helminth eggs, followed by potentially lethal portal hypertension. In this study, we characterized hepatic and splenic features of a murine model of intestinal schistosomiasis using in vivo magnetic resonance imaging (MRI) and evaluated the transverse relaxation time T₂ as a non-invasive imaging biomarker for monitoring hepatic fibrogenesis.Conclusions/SignificanceOur multiparametric MRI approach confirms that this murine model replicates hepatic and splenic manifestations of human intestinal schistosomiasis. Quantitative T₂ mapping proved sensitive to assess liver fibrogenesis non-invasively and may therefore constitute an objective imaging biomarker for treatment monitoring in diseases involving hepatic fibrosis.     

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.     

Effects of Murine Norovirus Infection on a Mouse Model of Diet-Induced Obesity and Insulin Resistance

Murine norovirus (MNV) is prevalent in SPF mouse facilities in the United States, and we currently lack sufficient data to determine whether it should be eliminated. It is generally accepted that the virus does not cause clinical symptoms in immunocompetent mice. However, we previously reported that MNV infection alters the phenotype of a mouse model of bacteria-induced inflammatory bowel disease in part through its effects on dendritic cells. The tropism of MNV toward macrophages and dendritic cells makes MNV a potential intercurrent variable in murine models of macrophage-driven inflammatory diseases, such as obesity, insulin resistance, and atherosclerosis. Therefore, we determined whether MNV infection altered obesity and insulin resistance phenotypes in C57BL/6 mice, a widely used model of diet-induced obesity. We found that MNV did not alter weight gain, food intake, and glucose metabolism in this model, but it did induce subtle changes in lymphoid tissue. Further studies using other models of metabolic diseases are needed to provide additional information on the potential role this ‘subclinical’ virus might have on disease progression in mouse models of inflammatory diseases.Abbreviations: HFD, high-fat diet; IPGTT, intraperitoneal glucose tolerance test; IPITT, intraperitoneal insulin tolerance test; MLN, mesenteric lymph node; MNV, murine norovirusMurine norovirus (MNV) is endemic in many SPF mouse colonies across North America,⁵ creating considerable potential for this virus to interfere with mouse models of human diseases. In addition, the presence of MNV in some mouse colonies and not in others may help explain phenotypic variability in mouse models across institutions. This virus is related to the human Norwalk virus that causes gastrointestinal inflammation in humans. Although MNV does not cause any overt illness in immunocompetent mice, significant inflammation and mortality can be induced in mice with abnormal innate immunity.⁷ Previously, we investigated the influence of MNV on the development of bacteria-induced inflammatory bowel disease in FVB.129P2-PAbcb1atm1Bor (Mdr1a^−/−) mice.⁸ We found that infection with MNV accelerated the progression of inflammatory bowel disease in this mouse model when mice were coinfected with Helicobacter bilis. In addition, infection with MNV alone altered the immune response, probably through changes in dendritic cells.⁸ These findings suggest that MNV may induce subtle changes in immune responses even in immunocompetent mice, given that MNV is known to preferentially infect macrophages and dendritic cells.²²Obesity has been defined as a disease of chronic inflammation, and in recent years, the prominent role that macrophages play in this process has been recognized.^9,10,21,24 Obesity is a risk factor for various chronic diseases that share inflammation as a critical component of the disease process, such as metabolic syndrome, diabetes, and atherosclerosis.³ Because MNV has tropism for macrophages, we wished to determine whether MNV infection influences the development of obesity and insulin resistance in a widely used animal model of diet-induced obesity. C57BL/6 mice are the most frequently used ‘wild-type’ strain and are prone to develop insulin resistance as obesity develops during high-fat feeding.¹ We hypothesized that MNV may accelerate inflammation by stimulating macrophage accumulation in adipose tissue, resulting in a more severe obesity or insulin resistance phenotype when mice are fed a high-fat diet.     

Proteomic Analysis of Mn-induced Resistance to Powdery Mildew in Grapevine

Previous studies documented that metal hyperaccumulation armours plants with direct defences against pathogens. In the present study, it was found that high leaf Mn concentrations (<2500 μg g(-1)) induced grapevine resistance to powdery mildew [Uncinula necator (Schw.) Burr]. Manganese delayed pathogen spreading after powdery mildew (PM) inoculation, but did not directly inhibit pathogen growth on a long-term basis. It was postulated that the grapevine resistance resulted from the induction of protective mechanisms in planta. To test this hypothesis, the proteome profile was analysed by Difference Gel Electrophoresis (DIGE) methods to identify proteins that are putatively involved in pathogen resistance. A high Mn concentration caused little oxidative pressure in grapevine, but oxidative stress was deeply enhanced by PM stress. Except for a few proteins that were related to oxidative pressure and proteins specially regulated by Mn or PM, most of the detected proteins exhibited similar changes under excess Mn stress and under PM stress, suggesting that similar signalling processes mediate the responses to the two stresses. As well as PM stress, high leaf Mn concentration significantly enhanced salicylic acid concentration and increased the expression of proteins involved in ethylene and jasmonic acid synthesis. The proteins related to pathogen resistance were also enhanced by excess Mn, including a PR-like protein, an NBS-LRR analogue, and a JOSL protein, and this was accompanied by the increased activity of phenylalanine ammonia lyase. It was concluded that high leaf Mn concentration triggered protective mechanisms against pathogens in grapevine.     

Proteomic Analysis of Adult Ascaris suum Fluid Compartments and Secretory Products

Background

Strategies employed by parasites to establish infections are poorly understood. The host-parasite interface is maintained through a molecular dialog that, among other roles, protects parasites from host immune responses. Parasite excretory/secretory products (ESP) play major roles in this process. Understanding the biology of protein secretion by parasites and their associated functional processes will enhance our understanding of the roles of ESP in host-parasite interactions.

Methodology/Principal Findings

ESP was collected after culturing 10 adult female Ascaris suum. Perienteric fluid (PE) and uterine fluid (UF) were collected directly from adult females by dissection. Using SDS-PAGE coupled with LC-MS/MS, we identified 175, 308 and 274 proteins in ESP, PE and UF, respectively. Although many proteins were shared among the samples, the protein composition of ESP was distinct from PE and UF, whereas PE and UF were highly similar. The distribution of gene ontology (GO) terms for proteins in ESP, PE and UF supports this claim. Comparison of ESP composition in A. suum, Brugia malayi and Heligmosoides polygyrus showed that proteins found in UF were also secreted by males and by larval stages of other species, suggesting that multiple routes of secretion may be used for homologous proteins. ESP composition of nematodes is both phylogeny- and niche-dependent.

Conclusions/Significance

Analysis of the protein composition of A. suum ESP and UF leads to the conclusion that the excretory-secretory apparatus and uterus are separate routes for protein release. Proteins detected in ESP have distinct patterns of biological functions compared to those in UF. PE is likely to serve as the source of the majority of proteins in UF. This analysis expands our knowledge of the biology of protein secretion from nematodes and will inform new studies on the function of secreted proteins in the orchestration of host-parasite interactions.     

A Metabolomic Analysis of Two Intravenous Lipid Emulsions in a Murine Model

Background

Parenteral nutrition (PN), including intravenous lipid administration, is a life-saving therapy but can be complicated by cholestasis and liver disease. The administration of intravenous soy bean oil (SO) has been associated with the development of liver disease, while the administration of intravenous fish oil (FO) has been associated with the resolution of liver disease. The biochemical mechanism of this differential effect is unclear. This study compares SO and FO lipid emulsions in a murine model of hepatic steatosis, one of the first hits in PN-associated liver disease.

Methods

We established a murine model of hepatic steatosis in which liver injury is induced by orally feeding mice a PN solution. C57BL/6J mice were randomized to receive PN alone (a high carbohydrate diet (HCD)), PN plus intravenous FO (Omegaven®; Fresenius Kabi AG, Bad Homburg VDH, Germany), PN plus intravenous SO (Intralipid®; Fresenius Kabi AG, Bad Homburg v.d.H., Germany, for Baxter Healthcare, Deerfield, IL), or a chow diet. After 19 days, liver tissue was harvested from all animals and subjected to metabolomic profiling.

Results

The administration of an oral HCD without lipid induced profound hepatic steatosis. SO was associated with macro- and microvesicular hepatic steatosis, while FO largely prevented the development of steatosis. 321 detectable compounds were identified in the metabolomic analysis. HCD induced de novo fatty acid synthesis and oxidative stress. Both FO and SO relieved some of the metabolic shift towards de novo lipogenesis, but FO offered additional advantages in terms of lipid peroxidation and the generation of inflammatory precursors.

Conclusions

Improved lipid metabolism combined with reduced oxidative stress may explain the protective effect offered by intravenous FO in vivo.     

An Experimental Investigation of the Resistance of Model Root Systems to Uprooting

The architecture of a tree root system may influence its abilityto withstand uprooting by wind loading. To determine how theroot branching pattern may alter the anchorage efficiency ofa tree, artificial model root systems with different topologiesand branching angles were built. The root systems were embeddedat various depths in wet sand and the pull-out resistance measured.A model to predict the uprooting resistance from the data collectedwas designed, allowing predictions of anchorage strength withregards to architecture. The dominant factors influencing pull-outresistance were the depth and length of roots in the soil. Themost efficient type of branching pattern predicted by the programwas one with an increased number of roots deep in the soil.The optimum branching angle most likely to resist pull-out isa vertical angle of 90° between a lateral and the main axis.The predicted mechanically optimal radial angle between a lateralbranch and its daughter is between 0 and 20°. Values ofbranching angle are compared with those measured in real woodyroot systems of European larch and Sitka spruce. Root architecture; root anchorage; pull-out resistance; windthrow; Picea sitchensis ; Larix decidua     

Proteomic Characterization of Acyclovir-Induced Nephrotoxicity in a Mouse Model

Acyclovir (ACV) is an effective and widely used antiviral agent. However, its clinical application is limited by severe nephrotoxicity. We assessed ACV-induced nephrotoxicity and identified the differentially expressed proteins using mass spectrometry-based proteomic analysis. In total, 30 ICR mice were intraperitoneally administrated ACV (150 or 600 mg/kg per day) for 9 days. After administration of ACV, levels of serum creatinine and urea nitrogen increased significantly. In addition, mouse kidneys exhibited histopathological changes and reduced expression levels of vascular endothelial growth factor (VEGF) and its receptor VEGFR2. In the proteomic analysis, more than 1,000 proteins were separated by two-dimensional polyacrylamide gel electrophoresis, and a total of 20 proteins were up- or down-regulated in the ACV group compared with the saline group. Among these, six proteins (MHC class II antigen, glyoxalase 1, peroxiredoxin 1, αB-crystallin, fibroblast growth factor receptor 1-IIIb, and cytochrome c oxidase subunit Vb) were identified in association with ACV-induced nephrotoxicity. These findings were confirmed by Western blotting analysis. The differential expression levels of α-BC, Prx1, Glo I and CcO Vb suggest that oxidative damage and mitochondrial injury may be involved in ACV-induced nephrotoxicity. Furthermore, VEGF and FGF may play a role in tissue repair and the restoration process following ACV nephrotoxicity.