1. Bradford A. Woodworth, MD
Characterization of Primary Rat Nasal Epithelial Cultures in CFTR-/- Rats as a Model for CF Sinus Disease
Kiranya E. Tipirneni, MD
Do-Yeon Cho, MD
Daniel F. Skinner, BS
Shaoyan Zhang, PhD
Calvin Mackey, BS
Dong-Jin Lim, PhD
Bradford A. Woodworth, MD
Good afternoon, I’m Kiranya Tipirneni, a post-doctoral research fellow at the University of Alabama at Birmingham. I want to thank the Triologic Society for allowing me to here and present this research with you all today.

2. Disclosures Bradford A. Woodworth, M.D.
Consultant for Olympus, Cook Medical, Smith and Nephew
Grant support from Cook Medical
National Institutes of Health (NIH)
National Heart, Lung, and Blood Institute (1 R01 HL ) to B.A.W.
National Institute of Diabetes and Digestive and Kidney Diseases (5P30DK , CF Research Center Pilot Award) to B.A.W.
National Cancer Institute (T32CA091078) to K.E.T.
This research was funded through the NIH by the National Heart, Lung, and Blood Institute and the NIDDK.

3. Background
Cystic fibrosis (CF) is the most common lethal genetic disease among Caucasians
The majority of patients develop CRS
Animal studies have been paramount in contributing to our understanding of CF
Pharmaceutical testing
Gene modification studies
First, I’ll begin with a very brief background about cystic fibrosis.
CF is the most common lethal genetic disease among Caucasians, with the majority of patients developing CRS.
Currently, animal studies have been paramount in contributing to our understanding of the disease, particularly in regards to:
Pharmaceutical testing
Gene modification studies, and
in vivo replication of CF pathogenesis
So, as you can imagine, development of a suitable CF animal model continues to be of significant interest

4. Current CF Animal Models
Currently, a number of CF animal models exist. These include the:
CF mouse,
Pig,
Ferret, and, more recently,
The CF rat.

5. Advantages to CF Rat Model
Larger size (relative to mice) allows for:
- Evaluation of multiple organs
Surgical procedures
Brief gestational period (21-23 days) & rapid maturation (8 weeks)
Rats are traditional animal model for pharmacology/toxicology studies
Cost
Several advantages to CF rat model:
Relative to mice, rats are significantly larger; thus, permitting sampling of larger tissue samples and surgical interventions not possible in smaller rodents
Unlike pig and ferret models, rats have a brief gestational period followed by swift sexual maturation, which facilitates rapid colony propagation, breeding and longitudinal studies
Furthermore, rats are also widely used for pharmacology and toxicity studies due to favorable pharmacokinetic and biodistribution profiles
And lastly, they are significantly less costly in comparison to ferrets and pigs

6. Rat Nasal Septal Epithelium (RNSE)
Primary RNSE cells cultured at an air-liquid interface on semipermeable supports
Scanning electron microscopy (SEM)
Histology
Ion transport studies
RT-PCR
In this study, we aimed to develop primary RNSE cultures and evaluate their utility as a model for investigating sinonasal transepithelial transport and CFTR function.
Nasal septa of wild type and knockout rats were collected and cultured on semipermeable supports at an ALI.
We evaluated RNSE cultured monolayers using SEM, histology, Ussing chamber analysis for pharmacologic manipulation and measurement of ion transport, and RT-PCR to evaluate relative CFTR gene expression.

7. Species Comparison with
Mouse and Human NSE
Primary MNSE and HNSE cells cultured at an air-liquid interface on semipermeable supports
Ion transport studies
RT-PCR
Additionally, sinonasal epithelia from mice and humans were cultured at an ALI for comparing ion transport and gene expression between the different species.

8. Ciliary differentiation visualized with SEM
CFTR+/+ 5x
25x
CFTR-/-
By day 14, cultured monolayers demonstrated complete differentiation
We identified robust ciliary beating with 80 to 90% ciliation throughout the apical monolayer surface, shown here at low and high magnification. 5x
25x

9. CF sinus epithelia have increased submucosal glands
Intraepithelial Mucus/ Basement Membrane
CFTR+/+
6.1 ± 0.5 µm2/ mm
CFTR-/-
8.7± 0.9 µm2/ mm
CFTR+/+
CFTR-/-
We then examined rat maxillary sinonasal epithelia for submucosal gland density with Alcian blue-periodic acid Schiff staining
While we found no evidence of infection, we did find that KO rats demonstrated an increased number of submucosal glands with an average of 9 per mm basement membrane vs 6 glands in the WT model
Glands shown in black arrows.

10. RNSE ISC measurements indicate CFTR-/- rats exhibit a robust bioelectric phenotype* * *
Next, we performed ion transport studies of WT and KO rats after pharmacologic manipulation with:
Amiloride—an epithelial Na+ channel inhibitor (ENaC)
Forskolin—CFTR activator that acts via cAMP-dependent pathway
INH-172—highly specific inhibitor of CFTR that blocks CFTR-dependent chloride channel; and
UTP—stimulator of Ca-activated Cl- channels (i.e. TMEM16A)
On the right, we have Ussing chamber short circuit current tracings where positive deflection represents anion moving from serosal to mucosal direction
On the left, which summarizes our short circuit current measurements, you can clearly see WT rats exhibit a significantly more robust forskolin response and is consistent with the presence of CFTR
On the other hand, KO rats demonstrate a significantly increased caclium-activated Cl- secretion with UTP-stimulated current
which suggests that alternative calcium-activated Cl- pathways are abundant and possibly serve to ameliorate Cl- ion imbalance in CFTR deficient states.
Which possibly can be account for lack of sinus disease in these models
*p<0.05

11. RNSE have increased ion transport phenotype compared to MNSE and HNSE* *
CFTR+/+ * * *
*p<0.05 * *
CFTR-/-
For comparison, we performed similar pharmacologic manipulation and ion transport studies using mouse and human NSE cultures.
Overall, we found that both WT and KO RNSE exhibit a more robust ion transport phenotype as compared to that observed in mice and humans.
Use mouse cursor to point to WT current tracing

12. RNSE have similar CFTR expression as compared to MNSE and HNSE
Lastly, we performed RT-PCR to evaluate relative amounts of CFTR expression in WT cultures derived from rats, mice, and humans.
Our results indicate that there is comparable CFTR expression among these species
Thus, indicating that the more robust ion transport phenotype exhibited by rats is not simply due to increased expression of CFTR channels

13. Summary of Rat Upper Airway
CF rat sinonasal epithelia have increased submucosal glands
KO rats exhibit robust bioelectric CF phenotype as indicated by ISC measurements of NSE
UTP-stimulated ISC measurements in KO rats suggest presence of abundant alternative Cl- pathways
RNSE have a more robust ion transport phenotype than that seen in mice or humans
So, in summary:
CF rat sinonasal epithelia demonstrate increased number of submucosal glands but no histological evidence of infection
Short circuit current measurements of KO RNSE cultures reflect a robust bioelectric CF phenotype
The robust UTP-stimulated current seen in KO rats suggests the presence of abundant alternative Cl- transport pathways, which may be responsible for lack of sinus disease observed in these models
In comparison to mice and humans, RNSE exhibit increased ion transport phenotype in all pharmacologic manipulation studies (except UTP in WT)

14. Conclusions
Provides a means for examining in vitro pharmacologic manipulation
Useful resource for longitudinal studies (intrauterine through adulthood) for:
Sinus development
Electrophysiology
Other endpoints relevant to disease mechanism
So what do all these findings mean?
In conclusion, RNSE cultures represent a good model for in vitro pharmacologic manipulation studies.
In fact, the robust ion transport phenotype of RNSE cultures suggest rats are more responsive and susceptible to ion transport modulators and may represent a better model for in vitro ion transport studies when compared to mice.
Lastly, they may be useful for longitudinal studies of sinus development, electrophysiology, and other pathophysiologic processes of CF.

15. Acknowledgements University of Alabama at Birmingham Woodworth Lab
Bradford A. Woodworth, MD
Do-Yeon Cho, MD
Daniel F. Skinner, BS
Shaoyan Zhang, PhD
Calvin Mackey
Dong-Jin Lim, PhD
UAB Animal Models Core Facility
Gregory Fleming James CF Research Center
Thank you to the Triological Society for allowing me to be here and special thanks to Dr. Woodworth and everyone in Woodworth lab giving me the opportunity to present this data on their behalf. I will now take any questions.