Inhalation exposure systems for the development of rodent models of sulfur mustard-induced pulmonary injury

Abstract

Sulfur mustard (SM) is a chemical threat agent for which its effects have no current treatment. Due to the ease of synthesis and dispersal of this material, the need to develop therapeutics is evident. The present manuscript details the techniques used to develop SM laboratory exposure systems for the development of animal models of pulmonary injury. These models are critical for evaluating SM injury and developing therapeutics against that injury. Iterative trials were conducted to optimize a lung injury model. The resulting pathology was used as a guide, with a goal of effecting homogeneous and diffuse lung injury comparable to that of human injury. Inhalation exposures were conducted by either nose-only inhalation or intubated inhalation. The exposures were conducted to either directly vaporized SM or SM that was nebulized from an ethanol solution. Inhalation of SM by nose-only inhalation resulted in severe nasal epithelial degeneration and minimal lung injury. The reactivity of SM did not permit it to transit past the upper airways to promote lower airway injury. Intratracheal inhalation of SM vapors at a concentration of 5400 mg x min/m(3) resulted in homogeneous lung injury with no nasal degeneration.

FIGURE 1

Sulfur mustard exposure suite floor plan

FIGURE 2

J-tube for sulfur mustard vapor generation. Neat sulfur mustard is introduced into the stainless steel J-tube via syringe pump. The J-tube is packed with glass beads and wrapped with heat tape to control the temperature of vaporization. Vaporization is conducted under nitrogen at a flow of 2 L/min.

FIGURE 3

Inhalation exposure schematic showing a nose-only inhalation system that precedes charcoal/HEPA filters that are used to remove SM from the effluent. Sample ports on either side of the exhaust filter enable evaluation of scrubbing efficiency.

FIGURE 4

Intubation inhalation exposure system schematic. Vapors are introduced to a central distribution plenum and then diverted to individual exposure plenums that are connected directly to an intubation tube. Rodents breathe on demand from the exposure plenum. Exhaust from the exposure plenum is controlled at 700 ml/min by a computer controlled mass flow controller (MFC). SM is removed from the exhaust by a HEPA and charcoal filtration system.

FIGURE 5

Exposure sled schematic illustrating the intubation sled that is used to deliver SM directly to the intubated rat. SM vapors traverse through the exposure plenum at 700 ml/min, and the rat breathes from the plenum on demand. Oxygen and SM concentration are measured directly from the exposure plenum.

FIGURE 6

Particle size distribution of nebulized SM ethanol solutions. SM was dissolved in ethanol and aerosolized in a Swirler nebulizer that is designed to deliver submicron aerosols. Particle size, measured by an aerodynamic particle sizer, confirmed that the nebulized material was submicron.

FIGURE 7

Concentration of SM over 80 min, illustrating the rise to target concentration and stability of concentration in the intubation inhalation system.

FIGURE 8

Transverse sections of the nasal vestibule at the level of the labial surface of the incisors of a control rat (A) and a rat exposed to SM vapor by nose-only exposure (50 mg/m³ for 60 min) at 8 days post exposure (B). Typically, SM caused severe necrosis and degeneration of the nasal mucosa, particularly in the rostral aspects of the nasal passages. Often, passages were partially occluded with necrotic cell debris and a protein-rich inflammatory exudate containing large numbers of neutrophils. Bars = 250 μm.

FIGURE 9

Lungs depicting the axial airways from rats exposed to filtered air (control, A) or to SM vapor by nose-only exposure (50 mg/m³ for 60 min) at 6 days post exposure (B). Airway epithelial injury is not apparent in (B). Bars = 250 μm.

FIGURE 10

(A) Proximal axial airway of a rat exposed to SM vapor via an intratracheal tube (180 mg/m³ for 21 min) at 4 days post exposure. Necrosis and hemorrhage effaces the mucosal epithelium (arrows) while fibrin, blood, and necrotic cell debris nearly completely occlude the lumen (arrowheads). (B) Proximal axial airway in the lung of a rat exposed to SM vapor via an intratracheal tube (139 mg/m³ for 60 min) at 1 day post exposure. Portions of the mucosal epithelium are sloughed (arrowheads), and hemorrhage is present in the submucosa (arrow). Bars = 100 μm.