Strip Meniscometry Tube in the Assessment of Tear Lactoferrin in Non-Obese Diabetic (NOD) Mice
Round 1
Reviewer 1 Report
Title: “Strip Meniscometry Tube in the Assessment of Tear Lactoferrin in Non-obese Diabetic (NOD) Mice”
Overall, the manuscript was well written, and all the references are in proper format. The abstract is clear and reflect the manuscript theme. Experimental part is well explained too. The conclusions are clear and in consistent with results.
But introduction is very short. This ection must be extended.
Taking into account the above-mentioned reasons, I suggest publication of the paper after minor revision.
Author Response
Thank you for this nice comment and the favorable view on the MS itself. We agreed with the reviewer that the introduction needs enrichment and it was rewritten as follows adding appropriate references:
” Sjögren’s syndrome (SS) is a multifactorial autoimmune disorder, mainly affecting the salivary and lacrimal glands, which is influenced by genetic as well as environmental factors that are not completely understood as yet. Although dry eyes and dry mouth characterize the disease, the expression of clinical spectrum is diverse, extending from a solitary organ-specific autoimmune exocrinopathy to a systemic disorder affecting several organs .While Sjögren’s syndrome is one of the most common autoimmune diseases, it has no specific and non-invasive diagnostic tests. Diagnosis is made by biopsy of the exocrine glands and blood tests for specific antibodies. Since ethical issues strongly hamper the collection and study of tissue or tear samples in humans in order to investigate the pathogenetic mechanisms, animal models of SS or dry eye disease help us to analyze many pathogenetic relations from which conclusions can be drawn for similar human diseases. NOD mice have been reported to have similar ocular surface disease and dry eye features to human ocular surface disease, including decreased tear volume, the loss of corneal barrier integrity (increased staining scores), reduced tear mucins, and increased lacrimal gland inflammatory infiltrates
, which led us to choose NOD mice to examine tear lactoferrin changes. Indeed, tear lactoferrin has been reported to be a good biomarker of dry eye disease in SS. The severity of ocular surface damage due to SS related dry eye largely depends on the tear secretory function of the lacrimal gland, and that the function of the lacrimal gland can be evaluated by determination of the level of tear lactoferrin.
Strip meniscometry tube (SMT), has been defined as a non-invasive tool for measuring the tear quantity in the human tear meniscus [1]. It has been shown to have a strong correlation with the Schirmer test, tear film break-up time (BUT), and ocular surface vital staining scores [2]. This new method of testing has not only been applied to the diagnosis of dry eye disease [3,4] but also for the assessment of the efficacy of punctal occlusion [5-7]. Recently, it has been shown to be a very useful tool in evaluating epiphora and related symptoms in humans [8].The applicability of SMT in tear volume measurements has also been shown in cats, dogs, mice and rabbits [9].
This new method has not been employed for the evaluation of tear protein levels in humans and in mice up until now.In this study, we examined the usefulness of SMT in tear lactoferrin concentration measurement in the NOD and WT mice.”
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The short article “Strip Meniscometry Tube in the Assessment of Tear Lactoferrin in Non-obese Diabetic (NOD) Mice” by Murat Dogru highlight the application of strip meniscometry tube (SMT) in the measurement of tear lactoferrin in dry eye animal model. There are few points need to be addressed before publication.
- The authors have chosen only male NOD mice why?
- Dry eye syndrome is a common complication of both type 1 and type 2 diabetes. NOD mice are considered as Type 1 diabetic model. Were the animals (NOD) used in this study diabetic or non-diabetic?
- What is the minimum volume of tear required for the measurement by SMT?
Author Response
The short article “Strip Meniscometry Tube in the Assessment of Tear Lactoferrin in Non-obese Diabetic (NOD) Mice” by Murat Dogru highlight the application of strip meniscometry tube (SMT) in the measurement of tear lactoferrin in dry eye animal model. There are few points need to be addressed before publication.
We sincerely thank this reviewer for such excellent comments which indeed increased the scientific value and understandability of the paper by the readers. Please find our responses below:
- The authors have chosen only male NOD mice why?
This was because Japan Clea, our mouse supplier had only male mice available at the time of conduct of the study. Moreover, previously we have conducted another study on a new tear and saliva biomarker(E-FABP) in NOD male mice ( in press with IJMS) so we had experience with the male NOD mice.
- Dry eye syndrome is a common complication of both type 1 and type 2 diabetes. NOD mice are considered as Type 1 diabetic model. Were the animals (NOD) used in this study diabetic or non-diabetic?
Please note that while the name is NOD, the full blown autoimmune disease and diabetes sets in around 12 weeks in female mice and much later in the male mice in our experience.However, the dry eye and LG changes appear much earlier and are believed to be an early manifestation of the full blown autoimmune disease in the NOD mice. The mice used in this study were tested by Clea Japan before shipment and did not yet display features of Type 1 diabetes. This is now clarified in methods as:” NODand WT mice were purchased from Japan Clea (Osaka, Japan) and had not yet developed type 1 diabetes at the time of the conduct of the current study.”
- What is the minimum volume of tear required for the measurement by SMT?
“Please note that three SM tear collections separated by one hour/eye were carried out which yielded between 1-3 microliters of tears which were diluted 10 times with the extracting solution. Thus 10-30 μL were available for ELISA assay.This is also now clarified in methods section as: SMT was gently immersed into the inferior TM of the mice with touch to the eyelid and ocular surface for 5 seconds. Three SMT tear collections were done on a single eye on the same day with an interval of one hour between the testings. The collections yielded 1-3 μL of tears/eye After collection of tears, the wetted part of the SMTs were cut with scissors and placed within a 1.5 mL Eppendorf tube to be stored in a -80 °C freezer until ELISA measurement. For ELISA, samples were allowed to defrost slowly on ice. 0.5M NaCl and 0.5% Tween80 in PBS solution was used to extract the collected tears from the SMTs. A 10 times dilution was done using this extracting solution based on the mm of wetting reading on the SMTs which yielded between 10-30 μL of diluted tears for the assays.”
Author Response File: Author Response.pdf
