In a recent ACT 2021 Talking Tox webinar titled “Safety of E-Cigarette Products – A Toxicological Challenge,” I provided scientific guidance on pathology endpoints for inhalation studies. During my segment, I discussed the special planning necessary to optimize histopathology results when studying test articles dosed via the inhalation route (nose-only, head, or whole-body exposure).

Throughout this special planning, there are three keys for achieving successful pathology endpoints in your inhalation toxicology study. You can watch the webinar for my detailed breakdown of what they are and how to accomplish them. Alternatively, keep reading for a recap of what I covered.

Key 1: STUDY DESIGN – CHOOSING THE RIGHT TISSUES TO EVALUATE

The first key to success happens during the study design phase. We want to collect tissues at necropsy that adequately capture the sites affected by inhaled drugs and particles, which may differ from those examined in studies using other dose routes. We also want to be mindful of species differences in tissue anatomy and sensitivity to insults. While mice and rats are often utilized in inhalation toxicology studies, others may incorporate dogs, monkeys, ferrets, rabbits, or hamsters.

Naturally, the upper and lower respiratory tract tissues should be collected and examined, including multiple sections of nasal cavity and larynx, trachea with carina (which is a lower section of trachea than is routinely examined in non-inhalation studies), and generally all lung lobes. Occasionally, with compounds suspected to affect the olfactory system, the cribriform plate of the ethmoid bone is also collected, through which olfactory nerves pass to reach the olfactory bulbs of the brain. Regional lymph nodes, including mediastinal and tracheobronchial nodes, are routinely evaluated in inhalation studies. Some nodes may be too small to reliably visualize at necropsy in mice and are captured fortuitously in lung sections or chunks of mediastinal fat submitted in the tissue block.

The stomach is not a site you might consider important in inhalation studies, but it can be a test article target when inhaled compounds are moved up the lung’s mucociliary apparatus and swallowed or because of grooming activities that remove test article from the fur after whole-body exposure. The non-glandular stomach of rats and mice is most often affected.

Systemic targets obviously vary by test article pharmacokinetics, and study design may incorporate a full tissue list unless it is an early pilot study with limited endpoints.

Remember that clinical pathology plays an important role in inhalation toxicology as well. Beyond hematology, clinical chemistry, and urinalysis, many inhalation studies incorporate bronchoalveolar lavage followed by cytologic analysis of the fluid and measurement of total protein, enzymes released from injured cells like lactate dehydrogenase and alkaline phosphatase, cytokines, and others. If lavage is performed on animals destined for histopathology, keep in mind that the lavage procedure itself may cause minor transient lung inflammation that resolves in two to four days.

Key 2: NECROPSY – WELL-PLANNED TISSUE COLLECTION AND FIXATION

The collection and handling of tissues at necropsy is a critical step, and that’s because we only get one chance to preserve them appropriately. A careful inspection of all tissues should be performed by the necropsy technician, including the oral cavity and pharynx – even if they will not be collected routinely (although tongue and pharynx may appear on a full tissue list). The lungs must be carefully removed intact without perforation, which will interfere with inflation with the fixative. Be sure to weigh the lungs before fixation (a necropsy error much more common than you would think!).

If a single animal will have lung tissue harvested and formalin-fixed for routine histopathology but also collected and frozen for molecular techniques, particle burden, or frozen tissue histologic staining, then you should conduct advanced planning. The best course of action is generally to include different study animals for pathology endpoints versus other assessments. However, in situations where a single rodent must be used for multiple purposes, there are varying opinions about which lobes to fix for histopathology and which to freeze or crush for other analyses. In our experience, the left lobe is most often fixed for histopathology with the right lobes used for the other analyses. The lobes destined for other analyses should be removed carefully from the lobes retained for histopathology, leaving as much of the lobar bronchi intact as possible to allow gentle insufflation of the pathology samples with formalin. Remember to retain the carina for histopathology as well.

The best practice for fixation is to inflate lungs slowly and consistently with a fixative – such as 10% neutral buffered formalin – under constant mild pressure using a gravity flow apparatus. While immersion fixation is simpler, it results in lung underinflation that confounds the detection of inflammatory cell infiltrates and other interstitial changes. Lungs can also be inflated with a syringe and manual pressure. However, take care not to use too much force; otherwise, you can create artifactual interstitial fluid that mimics edema.

Key 3: HISTOLOGY – CUSTOMIZED TO MEET INHALATION STUDY ENDPOINTS

After you have properly collected and fixed your pathology samples at necropsy, you’re ready to ensure meaningful outcomes during the histology step.

Because of unique anatomy and sensitivity, we want to be sure to capture specific sub-regions of tissues on the slides. For example, rodents are obligate nose breathers with complex nasal turbinates that account for their impressive sense of smell. Rodents also have cytochrome p450 enzyme activity in the nasal mucosa that can metabolize some test articles. We want to capture all four nasal epithelial zones on slides (squamous, transitional, respiratory, and olfactory). The nasopharynx near the Eustachian tubes is also sensitive to inhaled nasal toxicants in most species, where there is a transition from respiratory to squamous epithelium as it approaches the oropharynx.

The larynx is an area of high particle impact during inhalation and should be thoroughly examined in inhalation toxicology studies. In the rodent, the base of the epiglottis is extremely sensitive to inhaled toxicants due to high metabolic rate, rapid breathing, and a circuitous airflow pathway that increases toxicant deposition. Step sections are recommended to capture rodent larynx adequately on the slide. The dog and monkey are less susceptible to developing laryngeal lesions. When they occur, they are often on the vocal processes of the arytenoid cartilage because that’s where these larger species have a transition point from squamous to respiratory epithelium.

The carina, or bifurcation of trachea into main lobar bronchi, is also an area of high particle impact in all species and should be examined microscopically. The reason for its sensitivity is likely physical positioning as well as reduced efficiency of the mucociliary apparatus at airway branching points, and thus retention of test articles there. In addition to rodents, dogs and monkeys sporadically get loss of cilia, epithelial erosion, and hyperplasia at this point.

The main lung histology objective in inhalation studies is to capture both centroacinar (hilar) and peripheral lung fields on slides, which means sectioning the lobes roughly parallel to main bronchi to capture their branching. Many inhaled particles, especially when less than two microns in diameter, lead to changes at the level of the terminal and respiratory bronchioles and their adjacent alveoli.

When it comes to histologic stains, hematoxylin and eosin (H&E) staining will virtually always be involved because it offers excellent detail. Other special stains may be performed using the same formalin-fixed paraffin-embedded tissues by cutting additional slices from the block. Thus, while some special stains must be planned at study outset, others can be added later depending on the results of routine histopathology. Advanced planning is needed for frozen tissue stains, including Oil Red O. Tissues destined for frozen section histology are quickly frozen at the time of necropsy, often embedded in a cryomold containing OCT medium.

Immunohistochemical staining and immunofluorescence can often be performed on sections from the same paraffin block as routine slides were made. It’s a good idea not to over-fix tissues intended for these procedures, although an antigen retrieval step can often effectively unmask epitopes on the tissues and restore antibody binding. There are unlimited antibodies that can be used, depending on the protein of interest, including labels for native cell types, infiltrating leukocytes, and specific types of collagens.  

At the end of this process, a thorough collection of respiratory tissues is present on glass slides and ready for microscopic evaluation by a veterinary pathologist. Stained slides can also be digitally scanned and quantitatively analyzed by a human or AI program for parameters like alveolar or interstitial wall thickness or the distribution and intensity of collagen or cells labeled with various markers.

Conclusion

When you’re preparing for your next inhalation study, remember the 3 keys of achieving successful endpoints:

• Choosing the right tissues to evaluate (study design)
• Conducting well-planned tissue collection and fixation (necropsy)
• Customizing to meet your inhalation study endpoints (histology)

Refer to this blog post for guidance anytime or watch the webinar “Safety of E-Cigarette Products – A Toxicological Challenge”.

Additionally, here are some references you may find useful:

Chamanza R, Wright JA. A Review of the Comparative Anatomy, Histology, Physiology and Pathology of the Nasal Cavity of Rats, Mice, Dogs and Non-human Primates. Relevance to Inhalation Toxicology and Human Health Risk Assessment. J Comp Pathol. 2015 Nov;153(4):287-314.

Limjunyawong N, Mock J, Mitzner W. Instillation and Fixation Methods Useful in Mouse Lung Cancer Research. J Vis Exp. 2015 Aug 31;(102):e52964. This reference contains an embedded technical video by Johns Hopkins demonstrating mouse lung collection and fixative instillation beginning around 3:35.

Mowat V, Alexander DJ, Pilling AM. A Comparison of Rodent and Nonrodent Laryngeal and Tracheal Bifurcation Sensitivities in Inhalation Toxicity Studies and Their Relevance for Human Exposure. Toxicol Pathol. 2017 Jan;45(1):216-222.

National Toxicology Program Non-Neoplastic Lesion Atlas: https://ntp.niehs.nih.gov/nnl/index.htm

Registry of Industrial Toxicology Animal-data (RITA) Revised guides for organ sampling and trimming in rats and mice: https://reni.item.Fraunhofer.de/reni/trimming/

Renne R, Brix A, Harkema J, Herbert R, Kittel B, Lewis D, March T, Nagano K, Pino M, Rittinghausen S, Rosenbruch M, Tellier P, Wohrmann T. Proliferative and nonproliferative lesions of the rat and mouse respiratory tract. Toxicol Pathol. 2009 Dec;37(7 Suppl):5S-73S.

Renne RA, Gideon KM, Harbo SJ, Staska LM, Grumbein SL. Upper Respiratory Tract Lesions in Inhalation Toxicology. Toxicol Pathol. 2007;35(1):163-169.

Dr. Katie Knostman, Senior Director of General Toxicologic Pathology, StageBio