Eric D. Lombardini

Nonlesions, misdiagnoses, missed diagnoses, and other interpretive challenges in fish histopathology studies: a guide for investigators, authors, reviewers, and readers.

Differentiating salient histopathologic changes from normal anatomic features or tissue artifacts can be decidedly challenging, especially for the novice fish pathologist. As a consequence, findings of questionable accuracy may be reported inadvertently, and the potential negative impacts of publishing inaccurate histopathologic interpretations are not always fully appreciated. The objectives of this article are to illustrate a number of specific morphologic findings in commonly examined fish tissues (e.g., gills, liver, kidney, and gonads) that are frequently either misdiagnosed or underdiagnosed, and to address related issues involving the interpretation of histopathologic data. To enhance the utility of this article as a guide, photomicrographs of normal and abnormal specimens are presented. General recommendations for generating and publishing results from histopathology studies are additionally provided. It is hoped that the furnished information will be a useful resource for manuscript generation, by helping authors, reviewers, and readers to critically assess fish histopathologic data.

Hematopoietic Radiation Syndrome in the Gottingen Minipig

Abstract Additional large animal models for the acute radiation syndrome (ARS) would facilitate countermeasure development. We demonstrate here that Gottingen minipigs develop hematopoietic ARS symptoms similar to those observed in canines, non-human primates (NHPs) and humans. Dosimetry for whole-body γ irradiation (0.6 Gy/min) was performed using electronic paramagnetic resonance (EPR) with alanine; National Institute of Standards and Technology (NIST)-calibrated alanine pellets and water-filled Plexiglas phantoms were used. After irradiations of 1.6–2.0 Gy, blood pancytopenia was observed for several weeks, accompanied by the characteristic ARS stages: prodromal symptoms, latent period, illness and recovery or morbidity. Morbidity occurred between days 14 and 27, with a preliminary LD50/30 estimate between 1.7 and 1.9 Gy. The criterion of whether platelet counts were <200 × 103/µl 7 days postirradiation predicted whether animals would survive in 18 out of 20 cases. The degree of granulocytosis 3 h postirradiation was inversely correlated with survival. Animals euthanized based on preset morbidity criteria displayed signs of multi-organ dysfunction, including widespread internal hemorrhage and alterations in organ function reflected in blood chemistry. Circulating C-reactive protein (CRP), a marker for inflammation, became elevated within hours after irradiation, subsided after several days, and increased again after 14 days. The results support further development of the Gottingen minipig as a model for ARS.

The Gottingen Minipig Is a Model of the Hematopoietic Acute Radiation Syndrome: G-Colony Stimulating Factor Stimulates Hematopoiesis and Enhances Survival From Lethal Total-Body γ-Irradiation

PURPOSEnWe are characterizing the Gottingen minipig as an additional large animal model for advanced drug testing for the acute radiation syndrome (ARS) to enhance the discovery and development of novel radiation countermeasures. Among the advantages provided by this model, the similarities to human hematologic parameters and dynamics of cell loss/recovery after irradiation provide a convenient means to compare the efficacy of drugs known to affect bone marrow cellularity and hematopoiesis.nnnMETHODS AND MATERIALSnMale Gottingen minipigs, 4 to 5 months old and weighing 9 to 11 kg, were used for this study. We tested the standard off-label treatment for ARS, rhG-CSF (Neupogen, 10 μg/kg/day for 17 days), at the estimated LD70/30 total-body γ-irradiation (TBI) radiation dose for the hematopoietic syndrome, starting 24 hours after irradiation.nnnRESULTSnThe results indicated that granulocyte colony stimulating factor (G-CSF) enhanced survival, stimulated recovery from neutropenia, and induced mobilization of hematopoietic progenitor cells. In addition, the administration of G-CSF resulted in maturation of monocytes/macrophages.nnnCONCLUSIONSnThese results support continuing efforts toward validation of the minipig as a large animal model for advanced testing of radiation countermeasures and characterization of the pathophysiology of ARS, and they suggest that the efficacy of G-CSF in improving survival after total body irradiation may involve mechanisms other than increasing the numbers of circulating granulocytes.

A TPO receptor agonist, ALXN4100TPO, mitigates radiation-induced lethality and stimulates hematopoiesis in CD2F1 mice.

Abstract Thrombopoietin (TPO) receptor agonists lacking sequence homology to TPO were designed by grafting a known peptide sequence into the hinge and/or kappa constant regions of a human anti-anthrax antibody. Some of these proteins were equipotent to TPO in stimulating cMpl-r activity in vitro and in increasing platelet levels in vivo. ALXN4100TPO (4100TPO), the best agonist in this series with a Kd of 30 nM for cMpl-r, exhibited potent activity as a radiation countermeasure in CD2F1 mice exposed to lethal total-body radiation from a cobalt-60 γ-ray source. 4100TPO (2 mg/kg, s.c.) administered once either 24 h before or 6 h after TBI showed superior protection to five daily doses given before or after TBI. Prophylactic administration (69 to 94% survival) was superior to therapeutic schedules (60% survival). 4100TPO conferred a significant survival benefit (P < 0.01) when administered 4 days before or even 12 h after exposure and across a dose range of 0.1 to 8 mg/kg. The dose reduction factors (DRFs) with a single dose of 1 mg/kg 4100TPO 24 h before or 12 h after TBI were 1.32 and 1.11, respectively (P < 0.0001). Furthermore, 4100TPO increased bone marrow cellularity and megakaryocytic development and accelerated multi-lineage hematopoietic recovery in irradiated mice, demonstrating the potential of 4100TPO as both a protector and a mitigator in the event of a radiological incident.

Laparoscopic hysterectomy using various energy sources in swine: a histopathologic assessment

OBJECTIVEnAnalyze energy-induced damage to the swine vagina during laparoscopic hysterectomy.nnnSTUDY DESIGNnLaparoscopic colpotomy was performed in swine using ultrasonic, monopolar, and bipolar energy. Specimens (n = 22) from 13 swine were stained with hematoxylin and eosin and Massons trichrome for energy-related damage. The distal scalpel-cut margin was used as reference. Energy induced damage was assessed by gynecologic and veterinary pathologists blinded to energy source.nnnRESULTSnInjury was most apparent on Massons trichrome, demonstrating clear injury demarcation, allowing consistent, quantitative damage measurements. Mean injury was 0 ± 0 μM (scalpel, n = 22), 782 ± 359 μM (ultrasonic, n = 7), 2016 ± 1423 μM (monopolar, n = 8), and 3011 ± 1239 μM (bipolar, n = 7). Using scalpel as the reference, all were significant (P < .001).nnnCONCLUSIONnAll energy sources demonstrated tissue damage, with ultrasonic showing the least and bipolar the greatest. Further study of tissue damage relative to cuff closure at laparoscopic hysterectomy is warranted.

Anatomical and histological examination of the porcine vagina and supportive structures: in search of an ideal model for pelvic floor disorder evaluation and management.

Objective: The objective of the study was to examine the anatomy and histology of the swine vagina and adjacent supportive structures in comparison to human tissues to determine the potential utility of this model for pelvic floor disorder evaluation and management. Methods: This is a descriptive study of the gross anatomy and histology of the swine vagina, uterosacral ligament, cardinal ligament, and rectovaginal space. Tissue was collected from 6 different sites in each of the 6 animals, processed, and stained with hematoxylin-eosin, Masson trichrome, and van Gieson and evaluated by both gynecologic and veterinary pathologists. Results: Porcine tissues were similar to the human vagina and supporting structures. The origin, insertion, and course of the uterosacral and cardinal ligaments appeared similar to those in humans. Histologically, both the porcine and human vagina and rectum consist of a mucosal, muscular, and adventitial layers. The swine vaginal smooth muscle is arranged in an inner circular and outer longitudinal manner. Collagen, elastin, and smooth muscle were identified in 5 sites. Collagen was highest in the cardinal compared with the uterosacral ligament (P = 0.03), whereas elastin was highest in the uterosacral ligament. The vaginal epithelium measured approximately 40 &mgr;m at the vaginal cuff and 50 to 200 &mgr;m at anterior and posterior vagina. The swine vagina appeared thinner and contained less elastin. The rectovaginal region contained a smooth muscle layer leading to a thin adventitial layer. Conclusions: The swine vagina and adjacent supportive structures appear to be grossly and histologically similar to human vaginal anatomy, and these similarities could lead to further investigation of the porcine model in the study of pelvic support and support disorders.

Induction of Rhabdomyosarcoma by Embedded Military-Grade Tungsten/Nickel/Cobalt Not by Tungsten/Nickel/Iron in the B6C3F1 Mouse

Continued improvements in the ballistic properties of military munitions have led to metal formulations for which little are known about the long-term health effects. Previously we have shown that a military-grade tungsten alloy comprised of tungsten, nickel, and cobalt, when embedded into the leg muscle of F344 rats to simulate a fragment wound, induces highly aggressive metastatic rhabdomyosarcomas. An important follow-up when assessing a compound’s carcinogenic potential is to test it in a second rodent species. In this study, we assessed the health effects of embedded fragments of 2 military-grade tungsten alloys, tungsten/nickel/cobalt and tungsten/nickel/iron, in the B6C3F1 mouse. Implantation of tungsten/nickel/cobalt pellets into the quadriceps muscle resulted in the formation of a rhabdomyosarcoma around the pellet. Conversely, implantation of tungsten/nickel/iron did not result in tumor formation. Unlike what was seen in the rat model, the tumors induced by the tungsten/nickel/cobalt did not exhibit aggressive growth patterns and did not metastasize.

Multimodal noninvasive monitoring of soft tissue wound healing

Here we report results of non-invasive measurements of indirect markers of soft tissue healing of traumatic wounds in an observational swine study and describe the quantification of analog physiological signals. The primary purpose of the study was to measure bone healing of fractures with four different wound treatments. A second purpose was to quantify soft tissue wound healing by measuring the following indirect markers: (1) tissue oxygenation, (2) fluid content, and (3) blood flow, which were all measured by non-invasive modalities, measured with available devices. Tissue oxygenation was measured by near infrared spectroscopy; fluid content was measured by bipolar bio-impedance; and blood flow was measured by Doppler ultrasound. Immediately after comminuted femur fractures were produced in the right hind legs of thirty anesthetized female Yorkshire swine, one of four wound treatments was instilled into each wound. The four wound treatments were as follows: salmon fibrinogen/thrombin—nxa0=xa08; commercial bone filler matrix—nxa0=xa07; bovine collagen—nxa0=xa08; porcine fibrinogen/thrombin—nxa0=xa07. Fractures were stabilized with an external fixation device. Immediately following wound treatments, measurements were made of tissue oxygenation, fluid content and blood flow; these measurements were repeated weekly for 3xa0weeks after surgery. Analog signals of each modality were recorded on both the wounded (right) hind leg and the healthy (left) hind leg, for comparison purposes. Data were processed off-line. The mean values of 10-s periods were calculated for right–left leg comparison. ANOVA was applied for statistical analysis. Results of the bone healing studies are published separately (Rothwell et al. in J Spec Oper Med 13:7–18, 2013). For soft tissue wounds, healing did not differ significantly among the four wound treatments; however, regional oxygenation of wounds treated with salmon fibrinogen/thrombin showed slightly different time trends. Further studies are needed to establish standards for healthy wound healing and for detection of pathological alterations such as infection. Non-invasive measurement and quantification of indirect markers of soft tissue wound healing support the goals and principles of evidence-based medicine and show potential as easy to administer tools for clinicians and battlefield medical personnel to apply when procedures such as the PET scan are not available or affordable. The method we developed for storing analog physiological signals could be used for maintaining electronic health records, by incorporating vital signs such as ECG and EEG, etc.

Accelerated senescence in skin in a murine model of radiation-induced multi-organ injury

Accidental high-dose radiation exposures can lead to multi-organ injuries, including radiation dermatitis. The types of cellular damage leading to radiation dermatitis are not completely understood. To identify the cellular mechanisms that underlie radiation-induced skin injury in vivo, we evaluated the time-course of cellular effects of radiation (14, 16 or 17 Gy X-rays; 0.5 Gy/min) in the skin of C57BL/6 mice. Irradiation of 14 Gy induced mild inflammation, observed histologically, but no visible hair loss or erythema. However, 16 or 17 Gy radiation induced dry desquamation, erythema and mild ulceration, detectable within 14 days post-irradiation. Histological evaluation revealed inflammation with mast cell infiltration within 14xa0days. Fibrosis occurred 80 days following 17 Gy irradiation, with collagen deposition, admixed with neutrophilic dermatitis, and necrotic debris. We found that in cultures of normal human keratinocytes, exposure to 17.9 Gy irradiation caused the upregulation of p21/waf1, a marker of senescence. Using western blot analysis of 17.9 Gy-irradiated mice skin samples, we also detected a marker of accelerated senescence (p21/waf1) 7 days post-irradiation, and a marker of cellular apoptosis (activated caspase-3) at 30 days, both preceding histological evidence of inflammatory infiltrates. Immunohistochemistry revealed reduced epithelial stem cells from hair follicles 14-30 days post-irradiation. Furthermore, p21/waf1 expression was increased in the region of the hair follicle stem cells at 14 days post 17 Gy irradiation. These data indicate that radiation induces accelerated cellular senescence in the region of the stem cell population of the skin.

The Role of the Component Metals in the Toxicity of Military-Grade Tungsten Alloy

Tungsten-based composites have been recommended as a suitable replacement for depleted uranium. Unfortunately, one of these mixtures composed of tungsten (W), nickel (Ni) and cobalt (Co) induced rhabdomyosarcomas when implanted into the leg muscle of laboratory rats and mice to simulate a shrapnel wound. The question arose as to whether the neoplastic effect of the mixture could be solely attributed to one or more of the metal components. To investigate this possibility, pellets with one or two of the component metals replaced with an identical amount of the biologically-inert metal tantalum (Ta) were manufactured and implanted into the quadriceps of B6C3F1 mice. The mice were followed for two years to assess potential adverse health effects. Implantation with WTa, CoTa or WNiTa resulted in decreased survival, but not to the level reported for WNiCo. Sarcomas in the implanted muscle were found in 20% of the CoTa-implanted mice and 5% of the WTa- and WCoTa-implanted rats and mice, far below the 80% reported for WNiCo-implanted mice. The data obtained from this study suggested that no single metal is solely responsible for the neoplastic effects of WNiCo and that a synergistic effect of the three metals in tumor development was likely.