Frances M. Reid

Efficacy of Hydroxocobalamin for the Treatment of Acute Cyanide Poisoning in Adult Beagle Dogs

Introduction. The efficacy of hydroxocobalamin for acute cyanide poisoning was compared with that of saline vehicle in dogs. Methods. Anesthetized adult beagle dogs were administered potassium cyanide (0.4 mg/kg/min, IV) until 3 min after the onset of apnea. Hydroxocobalamin (75 mg/kg [n = 19] or 150 mg/kg [n = 18], IV) or saline vehicle [n = 17] was then infused over 7.5 min while animals were ventilated with 100% oxygen, which was stopped after 15 min. Results. In vehicle-treated animals cyanide produced deterioration that culminated in a moribund state requiring euthanasia within 4 h in 10 of 17 animals and in neurological deficits necessitating euthanasia within 2–4 d in an additional 4 animals (mortality rate 82%). Survival through 14 d was observed in 15 of 19 animals administered hydroxocobalamin 75 mg/kg (mortality rate 21%), and 18 of 18 administered hydroxocobalamin 150 mg/kg (mortality rate 0%). Conclusion. Hydroxocobalamin reversed cyanide toxicity and reduced mortality in a canine model.

A cutaneous full-thickness liquid sulfur mustard burn model in weanling swine: clinical pathology and urinary excretion of thiodiglycol.

Sulfur mustard (bis(2‐chloroethyl)sulfide, HD) is a well‐known blistering chemical warfare agent. We have developed a cutaneous full‐thickness HD burn model in weanling pigs for efficacy testing of candidate treatment regimens. This report addresses clinical pathology findings and the urinary excretion profile of a major HD metabolite (thiodiglycol, TDG) in this model. Six female Yorkshire pigs were exposed to HD liquid on the ventral surface for 2 h, generating six 3‐cm diameter full‐thickness dermal lesions per pig. Blood samples were collected throughout a 7‐day observation period for hematology and serum chemistry examinations. Urine was collected in metabolism cages. Routine urinalysis was performed and the urine analyzed for TDG using gas chromatography/mass spectrometry. Examination of clinical pathology parameters revealed subtle HD‐related changes that are suggestive of a mild hemolytic episode. No other signs of clinically significant systemic toxicities were noted, including bone marrow suppression. Thiodiglycol was detected at the earliest time point tested (6–8 h post‐exposure) at levels ranging from 0.66 to 4.98 μg ml−1 with a mean of 2.14 μg ml 2−1. Thiodiglycol concentrations were the highest for half of the animals at this earliest time point and at 24–48 h for the others. By the evening of day 3, the mean level had reached 50 ng ml−1. Mean levels remained 10–40 ng ml−1 for the remainder of the 7‐day observation period, with the highest individual concentration noted during this period of 132 ng ml−1. Our results are in general agreement with the TDG excretion profiles previously described for rodent models and humans. Urinary excretion of absorbed HD in our weanling pig wound healing model appears to follow the same pattern as is seen in other laboratory animals models. In general, urinary excretion of TDG appears to peak within the first 1–4 days following exposure, with detectable levels after 1 week. Relatively high urinary TDG levels may thus indicate agent exposure within the previous 96 h. Low levels significantly above natural background levels may indicate either exposure to low levels of agent or exposure that occurred more than 4 days prior to collection of the sample. Published in 2000 by John Wiley & Sons, Ltd.

Evaluation of immunogenicity and efficacy of anthrax vaccine adsorbed for postexposure prophylaxis.

ABSTRACT Antimicrobials administered postexposure can reduce the incidence or progression of anthrax disease, but they do not protect against the disease resulting from the germination of spores that may remain in the body after cessation of the antimicrobial regimen. Such additional protection may be achieved by postexposure vaccination; however, no anthrax vaccine is licensed for postexposure prophylaxis (PEP). In a rabbit PEP study, animals were subjected to lethal challenge with aerosolized Bacillus anthracis spores and then were treated with levofloxacin with or without concomitant intramuscular (i.m.) vaccination with anthrax vaccine adsorbed (AVA) (BioThrax; Emergent BioDefense Operations Lansing LLC, Lansing, MI), administered twice, 1 week apart. A significant increase in survival rates was observed among vaccinated animals compared to those treated with antibiotic alone. In preexposure prophylaxis studies in rabbits and nonhuman primates (NHPs), animals received two i.m. vaccinations 1 month apart and were challenged with aerosolized anthrax spores at day 70. Prechallenge toxin-neutralizing antibody (TNA) titers correlated with animal survival postchallenge and provided the means for deriving an antibody titer associated with a specific probability of survival in animals. In a clinical immunogenicity study, 82% of the subjects met or exceeded the prechallenge TNA value that was associated with a 70% probability of survival in rabbits and 88% probability of survival in NHPs, which was estimated based on the results of animal preexposure prophylaxis studies. The animal data provide initial information on protective antibody levels for anthrax, as well as support previous findings regarding the ability of AVA to provide added protection to B. anthracis-infected animals compared to antimicrobial treatment alone.

Sulfur mustard-induced skin burns in weanling swine evaluated clinically and histopathologically

Histopathological indicators and clinical observations were used to evaluate wound severity, depth and degree of healing on days 2 and 8 for full‐skin‐thickness sulfur‐mustard (HD)‐induced burns in weanling swine. Six female weanling swine were exposed for 2 h to 400 μl of HD at each of six dose sites on the hairless abdominal skin. Biopsy samples (8 mm) were taken from the periphery and from the center of the wound on day 2, and the wound was excised on day 8. Histopathological indicators evaluated were epidermal necrosis, follicular necrosis, dermal necrosis, vascular necrosis, depth of injury, ulceration (loss of epidermis), granulation tissue response, neovascularization, re‐epithelialization (hyperplasia) and completeness of healing. Wounds were more severe from anterior to posterior. Histopathological assessment of epidermal ulceration and necrosis of epidermis, dermis, basal epithelium, adnexal structures and subcutaneous tissue were useful indicators of wound development on day 2. Granulation tissue response (observed as early as day 8) and re‐epithelialization were good indicators of wound healing. Clinical evaluations were performed on day 2 prior to and after debriding, and on study day 8. Clinical observations on study day 2 were for wound size and for exudation, erythema, edema, necrosis and eschar. Clinical observations on study day 8 were for the previous parameters and for re‐epithelialization, granulation and infection. Wound size and severity increased from anterior to posterior position. Size, exudation and edema were useful indicators of wound development. These histological and clinical observation parameters will be used in future experiments to compare various treatments for HD‐induced burns. Copyright

Transcriptional changes in porcine skin at 7 days following sulfur mustard and thermal burn injury.

Severe cutaneous injuries continue to result from exposure to sulfur mustard [bis(2-chloroethyl)sulfide; HD] and thermal burns. Microarray analysis was utilized in this study to evaluate transcriptional changes in porcine skin assessing the underlying repair mechanisms of HD and thermal injury involved in wound healing. Four ventral abdominal sites on each of 4 weanling swine were exposed to 400 μL undiluted HD or a heated brass rod (70°C) for 8 minutes and 45–60 seconds, respectively. At 7 days postexposure, skin samples were excised and total RNA was isolated, labeled, and hybridized to Affymetrix GeneChip (Santa Clara, CA, USA) Porcine Genome Arrays (containing 20,201 genes). Based on the gene expression patterns in HD- and thermal-exposed skin at 7 days, the transcriptional profiles do not differ greatly. HD and thermal exposures promoted similar changes in transcription, where 270 and 283 transcripts were increased with HD and thermal exposures, respectively. Both exposures promoted decreases in 317 and 414 transcripts, respectively. Of the significantly increased transcripts, at least 77% were commonly expressed in both HD- and thermal-exposed skin, whereas at least 67% of decreased transcripts were common between both exposure types. Six of the top 10 biological functions were common to HD and thermal injury in which 9 canonical pathways were shared. The present study illustrates the similarities found between HD and thermal injury with respect to transcriptional response and wound healing and identifies specific genes (CXCL2, CXCR4, FGFR2, HMOX1, IGF1, PF4, PLAU, PLAUR, S100A8, SPP1, and TNC) that may be useful as potential therapeutic targets to promote improved wound healing.

Transcriptional responses associated with sulfur mustard and thermal burns in porcine skin

In military and civilian environments, serious cutaneous damage can result from thermal burns or exposure to the blistering agent sulfur mustard [bis (2-chloroethyl) sulfide; HD]. Similar therapies have historically been used to treat cutaneous thermal and HD injuries; however, the underlying molecular mechanisms of tissue damage and wound healing may differ between the types of burns. Using microarray analysis, this study assessed the transcriptional responses to cutaneous HD and thermal injury at 48 hours post-exposure to identify molecular networks and genes associated with each type of skin injury. Ventral abdominal sites on each of 4 weanling swine were exposed to 400 μl of undiluted HD or a heated brass rod (70°C) for 8 minutes and 45–60 seconds, respectively. At 48 hours post-exposure, total RNA was isolated from excised skin samples and hybridized to Affymetrix GeneChip Porcine Genome Arrays (containing 20,201 genes). Both HD and thermal exposure promoted significant transcriptional changes where 290 and 267 transcripts were increased and 197 and 707 transcripts were decreased with HD and thermal exposure, respectively. HD- and thermal-injured skin expressed 149 increased and 148 decreased common transcripts. Comparison of the 10 most significantly changed biological functions for HD and thermal exposures identified 7 overlapping functional groups. Canonical pathways analysis revealed 15 separate signaling pathways containing transcripts associated with both HD and thermal exposure. Within these pathways, 5 transcripts (CXCR4, FGFR2, HMOX1, IL1R1, and TLR4) were identified as known targets for existing phase II/III clinical trial or Food and Drug Administration (FDA)-approved drugs. This study is the first to directly assess transcriptional changes in porcine skin subjected to HD or thermal injury over the same time period.

Biomechanical monitoring of cutaneous sulfur mustard‐induced lesions in the weanling pig model for depth of injury

Background/purpose: A sulfur mustard (SM)‐induced cutaneous injury model was developed in weanling swine to evaluate the efficacy of candidate treatment regimens. Lesions were assessed clinically and histopathologically. Histopathologic evaluation of lesions was a subjective and invasive assessment. Biomechanical engineering methods offer an objective and less invasive method to evaluate lesions. The purpose of this study was to use biomechanical engineering instruments to assess SM‐induced lesions for depth of injury and to correlate those assessments with histopathology.

Gene expression analysis of bromine-induced burns in porcine skin.

Bromine is an industrial chemical that is irritating to the skin and causes cutaneous burns. An important factor in selecting or developing an effective treatment is to understand the underlying molecular mechanisms of tissue damage and wound healing. This study used a weanling swine burn model and microarray analysis to evaluate the effect of exposure length and sampling times on the transcriptional changes in response to cutaneous bromine injury. Ventral abdominal sites (N=4/treatment group) were exposed to 600microL undiluted bromine for 45 s or 8 min. At 24 h and 7d post-exposure, total RNA from skin samples was isolated, processed, and hybridized to Affymetrix GeneChip Porcine Genome Arrays. Expression analysis revealed that bromine exposure duration appeared to have less effect on the transcript changes than the sampling time. The percent transcripts changed at 24h were similar (30%) whether having a 45 s or 8 min bromine exposure; percent transcripts changed at 7d were also similar (62%) regardless of exposure length. However, only 13-14% of the transcripts were similar when comparing samples analyzed at 24h and 7d. Ingenuity Pathways Analysis (IPA) revealed six common biological functions among the top 10 functions of each experimental group, while canonical pathway analysis revealed 11 genes that were commonly shared among 24 significantly altered signaling pathways. Additionally, there were 11 signaling pathways in which there were no commonly shared transcripts. The present study is an initial assessment of the transcriptional responses to cutaneous bromine exposure identifying molecular networks and genes that could serve as targets for developing therapeutics for bromine-induced skin injury.

An assessment of transcriptional changes in porcine skin exposed to bromine vapor

Bromine is an industrial chemical that can cause severe cutaneous burns. This study was a preliminary investigation into the effect of cutaneous exposure to bromine vapor using a weanling swine burn model and microarray analysis. Ventral abdominal sites were exposed to a mean calculated bromine vapor concentration of 0.69 g L−1 for 10 or 20 min. At 48 h postexposure, total RNA from skin samples was isolated, processed, and hybridized to Affymetrix GeneChip Porcine Genome Arrays. Expression analysis revealed that bromine vapor exposure for 10 or 20 min promoted similar transcriptional changes in the number of significantly modulated probe sets. A minimum of 83% of the probe sets was similar for both exposure times. Ingenuity pathways analysis revealed eight common biological functions among the top 10 functions of each experimental group, in which 30 genes were commonly shared among 19 significantly altered signaling pathways. Transcripts encoding heme oxygenase 1, interleukin‐1β, interleukin 2 receptor gamma chain, and plasminogen activator inhibitor‐1 were identified as common potential therapeutic targets for Phase II/III clinical trial or FDA‐approved drugs. The present study is an initial assessment of the transcriptional responses to cutaneous bromine vapor exposure identifying molecular networks and genes that could serve as targets for developing therapeutics for bromine‐induced skin injury.

Effects of Three Anesthetic Regimens on Bioengineering Methods Conducted on Ventral Abdominal Skin of Weanling Swine

Background/Purpose: A weanling swine model was previously developed to study healing of cutaneous injuries induced by the blistering chemical warfare agent sulfur mustard. Noninvasive bioengineering methods are used in the model to monitor the progress of wound healing and evaluate the efficacy of treatments. It is necessary to anesthetize the animals to facilitate bioengineering data collection from ventral abdominal wound sites. As anesthetic agents affect cardiac output and deep vascular and cutaneous microcirculation in many species, there was a need to identify an anesthetic regimen with minimal effects on baseline measurements. The effects of three anesthetic regimens on reflectance colorimetry, transepidermal water loss, and laser Doppler perfusion imaging were studied in unexposed control animals. Methods: The following regimens were tested on six female Yorkshire swine (weanlings, 8–11 kg): repeated, separate intramuscular (i.m.) injections of ketamine HCl and xylazine HCl (K/X, at 20 ± 2 mg/kg and 2 ± 0.2 mg/kg, respectively), repeated i.m. injections of a tiletamine HCl/zolazepam HCl/xylazine HCl combination (T/X, at 2.2 mg/kg, 2.2 mg/kg, and 4.4 mg/kg, respectively), and the tiletamine HCl/zolazepam HCl/xylazine HCl combination as a preanesthetic and isoflurane inhalation to maintain anesthesia (T/X/Iso; dosage of tiletamine HCl/zolazepam HCl/xylazine HCl was the same as listed above, with 2.5–3.0% isoflurane in oxygen at an initial flow rate of 2 L/min, reduced to 1.0%–1.5% at 0.8–1.0 L/min for maintenance). Each regimen was administered in three iterations within a week (every other day), with a minimum 1‐week washout period between regimens. Results: The effect of the anesthetic regimens on bioengineering assessments of ventral abdominal skin was evaluated. For reflectance colorimetry, regimens T/X and K/X had a narrower range of readings over the three testing days than T/X/Iso. Either T/X or K/X was suitable, with T/X preferred because of a lesser blanching effect. T/X or T/X/Iso were preferred for transepidermal water loss readings, because overall they depressed transepidermal water loss rates less than did K/X. T/X, T/X/Iso, and K/X all affected cutaneous blood flow, with no clear preference. Conclusions: Overall, T/X produced the most consistent results with the fewest anesthetic effects.