Login processing...

Trial ends in Request Full Access Tell Your Colleague About Jove


A Minimally Invasive Method for Intratracheal Instillation of Drugs in Neonatal Rodents to Treat Lung Disease

Published: August 4, 2021 doi: 10.3791/61729


This technique of instilling drugs directly into the trachea of neonatal rodents is important in studying the impact of locally administered drugs or biologicals on neonatal lung diseases. Additionally, this method can also be used for inducing lung injury in animal models.


Treatment of neonatal rodent with drugs instilled directly into the trachea could serve as a valuable tool to study the impact of a locally administered drug. This has direct translational impact because surfactant and drugs are administered locally into the lungs. Though the literature has many publications describing minimally invasive transoral intubation of adult mice and rats in therapeutic experiments, this approach in neonatal rat pups is lacking. The small size of orotracheal region/pharynx in the pups makes visualization of laryngeal lumen (vocal cords) difficult, contributing to the variable success rate of intratracheal drug delivery. We hereby demonstrate effective oral intubation of neonatal rat pup - a technique that is non-traumatic and minimally-invasive, so that it can be used for serial administration of drugs. We used an operating otoscope with an illumination system and a magnifying lens to visualize the tracheal opening of the rat neonates. The drug is then instilled using a 1 mL syringe connected to a pipette tip. The accuracy of the delivery method was demonstrated using Evans blue dye administration. This method is easy to get trained in and could serve as an effective way to instill drugs into trachea. This method could also be used for administration of inoculum or agents to simulate disease conditions in animals and, also, for cell-based treatment strategies for various lung diseases.


or Start trial to access full content. Learn more about your institution’s access to JoVE content here

Neonates born prematurely have poorly developed lungs requiring many interventional therapies such as long-term ventilation. These interventions place the surviving neonates at a high risk of subsequent sequelae1. Experimental animal models serve as an important tool in simulating various disease conditions, studying the pathobiology of diseases, and evaluating therapeutic interventions. Even though a broad range of animal models from mice, rat, and rabbit to pre-term lambs and pigs are available, mice and rat are the most used.

The primary advantage of using mice and rats are the relatively short gestation period and reduced cost. They are also readily available, easy to maintain in disease-free environments, genetically homogeneous and have relatively less ethical concern2,3. Another major advantage of the rodent model is that at birth the neonatal pup is at late canalicular/early saccular stage of lung development which is morphologically equivalent to the lung of a 24-week preterm neonatal human infant going on to develop bronchopulmonary dysplasia4. In addition, as their lung development rapidly progresses to completion within the first 4 weeks of life, it is feasible to study the post-natal lung maturation in a reasonable time frame4. Despite these advantages, the small size of the mice and rat pups is a source of concern for various interventions, which compels most researchers to use adult animals rather than pups5. Neonatal lungs are in a developmental stage and the response of a neonate to an inciting agent differs from that of an adult. This makes it appropriate to use neonatal animal models to study human neonatal disease conditions.

There are different methods to administer drugs/ biological agents to the lung. This includes intranasal6,7 or intratracheal8,9,10 instillation as well as aerosol inhalation11,12. Each approach has its own technical challenges, advantages, as well as limitations13. Intratracheal route of administration of therapeutic agents is preferred to study the direct therapeutic impact in the organ bypassing the systemic effects. This route could also be used to study lung pathology caused by inciting agents. There are both invasive and minimally invasive techniques to do this and is easy to perform in adults. However, in pups, because of the small size of the animal, there are technical challenges associated with the intubation process. The current study presents a simple, consistent, non-surgical intratracheal instillation (ITI) method in rat pups that could be used to study the efficacy of various neonatal therapeutic interventions as well as to generate animal models simulating neonatal respiratory diseases.

Subscription Required. Please recommend JoVE to your librarian.


or Start trial to access full content. Learn more about your institution’s access to JoVE content here

All experiments were approved by the Institutional Animal Care and Use Committee (protocol # 2020-0035) at the Case Western Reserve University. All animals were treated in accordance with the NIH guidelines for the care and use of laboratory animals.

1. Animals

  1. Commercially obtain pregnant Sprague Dawley rats.
  2. Maintain animals at an approved veterinary facility with 14 h/10 h light-dark cycle and 45-60% relative humidity.

2. Preparation of test compound

  1. Use Evans blue dye as the test compound to assess the efficacy of the intratracheal instillation procedure.
  2. Prepare a 0.25% (w/v) solution of the dye in phosphate-buffered saline (pH 7.2) and filter sterilize using a 0.45 µm syringe filter.

3. Administration of anesthesia

  1. Anesthetize rat pups using gas anesthesia (3% isoflurane in 100% oxygen), using a modified delivery system adapted for small rat neonates.
  2. Check for the loss of tail and pedal reflexes and shallow breathing to ensure the proper depth of anesthesia for carrying out the procedure.

4. Intratracheal instillation (ITI)

  1. Use rat pups at post-natal day 5 (PN 5) for the ITI. Average weight of a PN 5 rat pup is 12 grams.
  2. Restrain the anesthetized rat pup on an inclined flat platform using laboratory labelling tape. The pup is restrained at an angle of about 45° in the supine position.
  3. Open the mouth of the neonate, and gently pull the tongue out to one side using a blunt forceps.
  4. Use a small otoscope speculum of 2 mm diameter connected to the otoscope to hold the tongue gently and for proper visualization of the larynx.
  5. Use the throat illuminator system i.e., the operating otoscope, and the magnifying lens for proper visualization of vocal cords (Figure 1).
  6. Position the animals at an angle of 45° in an inclined plane. The wired bar lids of mouse cages are used (Figure 2).
    NOTE: Positioning the animal at an angle of 45° provides better visualization of tracheal opening without the interference of the epiglottis.
  7. Take a long-angled pipette tip which is used for loading western blot gels. Cut the base of the pipette tip using a surgical blade so that it fits well into the tip of 1 cc syringe.
  8. Use the sterile 1 mL syringe fitted into a long-angled pipette tip to deliver 30-50 µL of the substance into the lung. Invert the syringe and aspirate nearly 0.9 cc air into the 1 mL syringe connected to the pipette tip followed by the dye or the substance to be delivered. This allows the air behind the dye to be pushed into the trachea after the dye is administered as shown in Figure 3. The intratracheal administration is achieved by visualizing the laryngeal lumen (vocal cords) and inserting the pipette tip fitted to a syringe into the tracheal lumen.
  9. Use the speculum of the otoscope to hold the tongue and expose the vocal cords. Speculum serves the role of the blade of a laryngoscope. Bend the pipette tip to an angle of 30° to facilitate easy introduction of the agent through the cone-shaped speculum into the tracheal opening.
  10. Introduce the pipette tip into the tracheal opening to the point of about 2 mm beyond the vocal cords. Push the piston of the syringe to administer the dye or the drug through the speculum of the operating otoscope as shown in Figure 3. The introduction of air into the lung soon after the administration of the agent prevents the substance from coming back to the laryngeal cavity.
  11. After administering the pup with the dye or normal saline, place the pups on an integrated circulating fluid heating pad (38°C) until their respiratory movements are regular. After complete recovery from anesthesia, reunite the pups with the dam.

5. Characterization of ITI delivery

  1. After ITI, euthanize the rat pups by giving excessive anesthesia (Ketamine 100 mg/kg and Xylazine 10 mg/kg) / thiopentone followed by exsanguination at an appropriate time post-administration. Euthanasia was performed as part of the experiment to collect lung tissue to demonstrate the efficacy.
  2. Secure the euthanized rat pup on a dissection board and wipe the chest and abdomen with 70% ethyl alcohol.
  3. For evaluating the distribution of the dye throughout the lung, remove the lungs from the animal using sterile technique and display the lungs as appropriate for imaging (Figure 4A,B).

Subscription Required. Please recommend JoVE to your librarian.

Representative Results

or Start trial to access full content. Learn more about your institution’s access to JoVE content here

The instillation of Evans blue revealed multifocal distribution of the dye involving all pulmonary lobes (Figure 4A,B). Our result as shown in Figure 4 demonstrates efficacy of distribution to all lobes. The picture is taken immediately after ITI of the dye into the trachea. 100% efficacy was achieved in instilling the dye into the trachea followed by its spread into all the lobes on both sides. It is expected that the dye would spread further within the lobule of the lung. With repeated administration, we have been able to ensure 100% success in delivering this to the lung to both lobes and all lobules. We have ensured that no dye reaches stomach or outside of lungs. This testifies the efficacy of technique as 100% administration into the lungs. The isoflurane anesthesia allowed faster recovery of the pups after the procedure.
Rat pups from day 5 tolerated this procedure and took less than 5 minutes to carry out following anesthesia. Some animals though developed transient apnea, regained the normal respiratory pattern in a few minutes.

Figure 1
Figure 1: Otoscope components. (A) power source 2.5 V (B) magnifying lens (C) transilluminator (D) speculum. Please click here to view a larger version of this figure.

Figure 2
Figure 2: The positioning of the animal. The positioning of the animals at an angle of 45° provided better visualization of tracheal opening without the interference of the epiglottis. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Intratracheal instillation. Visualization of the tracheal opening using otoscope/ throat illuminator system to achieve direct delivery to lungs. Please click here to view a larger version of this figure.

Figure 4
Figure 4: ITI instillation and Evans blue staining. (A) ITI instillation delivers the dye throughout the lungs. The dye can be seen distributed to both lobes of the lung as indicated by the black arrow. Absence of dye in the stomach confirms the success of the technique (red arrow). (B) Lungs from rat pups instilled with 50 µL of 0.25% Evans blue dye. Please click here to view a larger version of this figure.

Subscription Required. Please recommend JoVE to your librarian.


or Start trial to access full content. Learn more about your institution’s access to JoVE content here

Intratracheal instillation is an excellent method that offers several advantages over the existing methods for respiratory disease interventions as well as disease model development. It is a quick method and with experience, can be performed with an average speed of 2-3 minutes per animal. The key considerations for a successful intubation are proper sedation of the animal, it's correct positioning, especially the head, as well as accurate depth of placement/ size of the specula in the oropharynx. Proper sedation would allow sufficient working time for the operators, especially beginners. Positioning of the animal at a 45° angle is important for proper visualization of vocal cords. Placement of speculum at the right depth helps in retraction of the tongue throughout the procedure which again allows good visualization of the vocal cord. A team of two people can easily coordinate this work. One could coordinate the anesthesia and caging of animals while the other could deal with the instillation. The most technically challenging part of ITI is the correct intubation into the trachea. Success of the technique is confirmed by administration of dye following intubation. It is very important to confirm the initial step of correct intubation, as there is a good chance for the tubing to slip into esophagus resulting in the delivery of the substance into the stomach, rather than the lung.

The only part that one must be careful is the potential trauma associated with misintubation. One also must be very gentle and careful in order to avoid penetrating through the trachea or the tissue surrounding the vocal cords. It is also recommended not to conduct ITI if there have been 2 or 3 misses2.

There are different routes for administration of drugs/biological agents with each one having its own inherent advantages and disadvantages. Selection of a method is based principally on study objectives and nature of the intervention. Both intranasal instillation and aerosolization techniques deliver agents to the upper respiratory tract as well as the lungs. This benefits studies involving upper respiratory tract13,21 however, the delivery of a substance to lungs is unreliable. In addition, swallowing, sneezing and the varying breathing rates may lead to inconsistencies in the doses delivered. However, the physicochemical properties of some substances affect their efficient aerosolization15. Researchers use intratracheal inoculation to get around this problem, which regardless of particle size and viscosity, delivers inoculum/drugs directly into the lungs23.

The two main intratracheal delivery methods include transoral intratracheal14,15 and transtracheal instillation with or without tracheotomy16,17. ITI is a procedure where a wide range of treatment doses can be administered to a large number of animals quickly, once trained18. While transoral intratracheal instillation is routinely used in adult rats, the more invasive technique such as surgical incision was required in neonates16,19,20. Researchers still avoid the use of this transoral ITI technique in pups because of several reasons. The small size of the neonatal rodent makes the visualization of the laryngeal lumen difficult along with poor success in intubation. Also, the traditional metal laryngoscope used for ITI in adults cannot be used in neonate because of the small size of the oral cavity and the fragile mucosal tissues16,18,10. Smaller speculum and catheters are required to view the laryngeal cavity and deliver the therapeutics/ agents into the lung. The operator must be highly skilled to achieve this. Finally, recovery from anesthesia, hypothermia, maternal rejection, and cannibalism create additional problems for rat neonate recovery and survival21,22. Our study employed the use of gas anesthesia followed by recovery in heating pads and reuniting with lactating dams. This avoids problems associated with hypothermia, maternal rejection, or cannibalism. Many of the non-surgical intervention studies involve a blind intubation of the trachea through the oral cavity. This is especially not acceptable in the case of drug where the effect may be missed if it is wrongly instilled into esophagus. In this study, the tracheal opening is visualized using an otoscope and a slightly bent pipette tip is inserted directly into the trachea to deliver the substance, the dye in this case. Our technique demonstrates an effective way of administering the drug into the trachea of a small rat pup.

The process of ITI, is a reliable method when performed following meticulous training. Once trained it can be done rapidly and effectively as in adult rodents13,24,25. The correct endotracheal instillation can be confirmed by several methods including the dye or liquid movement in a tubing or syringe26,27,28. As it is possible to visualize the tracheal opening in this method, the misses are very less. Apnea was observed in a few pups immediately after ITI which was recovered spontaneously18,29. Using otoscope along with the smallest speculum served as a perfect fit for the small oral cavity of the neonatal rat18. The results of this study indicated that the substance can be consistently delivered to all the lobes of the lung as confirmed by the dye localization. This method would be of great significance in experimental studies in which neonatal rats are required to reliably mimic neonatal lung conditions30,31,32. This technique could also be used to carry out lung function studies33 as well as cell/ stem cell transplantation studies34,35,36 which currently employs surgical interventions and could be distressing to pups.

This technique also contributes to the principles of Refinement and Reduction in animal research. This method serves as an alternative to direct intratracheal injection with a needle which is a blind technique and is invasive as it pierces the trachea causing pain and bleeding. In complete contrast this technique serves to reduce pain while refining the introduction of a drug into the trachea, achieving immediate reduction of pain and suffering, and improvement of welfare of animals involved in research37. In addition, the administration of drug into trachea is directly visualized ensuring efficacy. Though the instillation of drug into trachea is widely practiced in larger animal our refinement to use this in a 5-day old rat pup is the innovation we would like to stress here.

This article offers a simple, minimally invasive, and reproducible method which could be used for administration of injurious agents in order to simulate pathological conditions as well as for local administration of drugs, antioxidants, cells/ stem cells for neonatal therapies.

Subscription Required. Please recommend JoVE to your librarian.


The authors have nothing to disclose.


This work was supported in part by R01HD090887-01A1 from NICHD to AH. The authors also acknowledge the facilities provided by Dr. Peter Mc Farlane's lab such as inhalation anesthesia/ heating pad system. Ms. Catherine Mayer's valuable assistance in setting up of the system is appreciated. No role was played by the funding body in the design of the study, collection, analysis and interpretation of data or in writing the manuscript.


Name Company Catalog Number Comments
Evans Blue dye Sigma-Aldrich, St Louis, MO, USA 314-13-6 Confirmation of drug administration into lungs
Ketamine Hydrochloride Hospira. Inc, Lake Forest, IL, USA Dispensed from Animal care facility For sedation
Operating Otoscope Welch Allyn, Hillrom, Chicago, IL, USA 21770- 3.5V For visualization of vocal cords
Otoscope Rechargeable Handle Welch Allyn, Hillrom, Chicago, IL, USA 71050-C
Pipette tip (Gel loading) Fisherbrand 02-707-139 Administering the drug
Platform for restraining (inclined plane) Animal care facility Dispensed from Animal care facility Wired roof of mice cage can be used
3M Micropore Surgical White Paper (sticking tape) 3M, St. Paul, MN, USA 1530-2
Luer Lock SyringeSyringes (1 ml) BD Franklin Lakes, NJ , USA NBD2515 Administering the drug
Xylazine Hospira. Inc, Lake Forest, IL, USA For sedation



  1. Prakash, Y. S. Pulmonary Cell Biology Lab: Neonatal Lung Disease. Mayo Clinic. Available from: https://www.mayo.edu/research/labs/pulmonary-cell-biology/projects/neonatal=lung-disease (2020).
  2. Martínez-Burnes, J., López, A., Lemke, K., Dobbin, G. Transoral intratracheal inoculation method for use with neonatal rats. Comparative Medicine. 51, (2), 134-137 (2001).
  3. Pinkerton, K. E., Crapo, J. D. Morphometry of the alveolar region of the lung. Toxicology of Inhaled Materials Handbook of Experimental Pharmacology. Witschi, H., Brain, J. D. 95, Springer, Berlin. 259-285 (1985).
  4. Nardiello, C., Mižíková, I., Morty, R. E. Looking ahead: where to next for animal models of bronchopulmonary dysplasia. Cell and Tissue Research. 367, (3), 457-468 (2017).
  5. Sugimoto, M., Ando, M., Senba, H., Tokuomi, H. Lung defenses in neonates: Effects of bronchial lavage fluids from adult and neonatal rabbits on superoxide production by their alveolar macrophages. Journal of the Reticuloendothelial Society. 27, (6), 595-606 (1980).
  6. Grayson, M. H., et al. Controls for lung dendritic cell maturation and migration during respiratory viral infection. Journal of Immunology. 179, (3), Baltimore, Md. 1438-1448 (2007).
  7. Moreira, A., et al. Intranasal delivery of human umbilical cord Wharton's jelly mesenchymal stromal cells restores lung alveolarization and vascularization in experimental bronchopulmonary dysplasia. Stem Cells Translational Medicine. 9, (2), 221-234 (2020).
  8. Bar-Haim, E., et al. Interrelationship between dendritic cell trafficking and Francisella tularensis dissemination following airway infection. PLoS Pathogens. 4, (11), 1000211 (2008).
  9. Linderholm, A. L., Franzi, L. M., Bein, K. J., Pinkerton, K. E., Last, J. A. A quantitative comparison of intranasal and intratracheal administration of coarse PM in the mouse. Integrative Pharmacology, Toxicology and Genotoxicology. 1, (1), 5-10 (2015).
  10. Guerra, K., et al. Intra-tracheal administration of a naked plasmid expressing stromal derived factor-1 improves lung structure in rodents with experimental bronchopulmonary dysplasia. Respiratory Research. 20, (1), 255 (2019).
  11. Thomas, R., et al. Influence of particle size on the pathology and efficacy of vaccination in a murine model of inhalational anthrax. Journal of Medical Microbiology. 59, Pt 12 1415-1427 (2010).
  12. Sakurai, R., et al. A combination of the aerosolized PPAR-γ agonist pioglitazone and a synthetic surfactant protein B peptide mimic prevents hyperoxia-induced neonatal lung injury in rats. Neonatology. 113, (4), 296-304 (2018).
  13. Rayamajhi, M., et al. Non-surgical intratracheal instillation of mice with analysis of lungs and lung draining lymph nodes by flow cytometry. Journal of Visualized Experiments. (51), e2702 (2011).
  14. Brain, J. D., Knudson, D. E., Sorokin, S. P., Davis, M. A. Pulmonary distribution of particles given by intratracheal instillation or by aerosol inhalation. Environmental Research. 11, (1), 13-33 (1976).
  15. Pritchard, J. N., et al. The distribution of dust in the rat lung following administration by inhalation and by single intratracheal instillation. Environmental Research. 36, (2), 268-297 (1985).
  16. Ruzinski, J. T., Skerrett, S. J., Chi, E. Y., Martin, T. R. Deposition of particles in the lungs of infant and adult rats after direct intratracheal administration. Laboratory Animal Science. 45, (2), 205-210 (1995).
  17. Sun, B., Curstedt, T., Song, G. W., Robertson, B. Surfactant improves lung function and morphology in newborn rabbits with meconium aspiration. Biology of the Neonate. 63, (2), 96-104 (1993).
  18. Nicholson, J. W., Kinkead, E. R. A simple device for intratracheal injections in rats. Laboratory Animal Science. 32, (5), 509-510 (1982).
  19. Carlon, M., et al. Efficient gene transfer into the mouse lung by fetal intratracheal injection of rAAV2/6.2. Molecular Therapy: The Journal of the American Society of Gene Therapy. 18, (12), 2130-2138 (2010).
  20. Chen, C. -M., Chen, Y. -J., Huang, Z. -H. Intratracheal Instillation of Stem Cells in Term Neonatal Rats. Journal of Visualized Experiments. (159), e61117 (2020).
  21. Reynolds, R. D. Preventing maternal cannibalism in rats. Science. 213, (4512), New York, N.Y. 1146 (1981).
  22. Park, C. M., Clegg, K. E., Harvey-Clark, C. J., Hollenberg, M. J. Improved techniques for successful neonatal rat surgery. Laboratory Animal Science. 42, (5), 508-513 (1992).
  23. Cleary, G. M., et al. Exudative lung injury is associated with decreased levels of surfactant proteins in a rat model of meconium aspiration. Pediatrics. 100, (6), 998-1003 (1997).
  24. Das, S., MacDonald, K., Chang, H. -Y. S., Mitzner, W. A simple method of mouse lung intubation. Journal of Visualized Experiments. (73), e50318 (2013).
  25. Oka, Y., et al. A reliable method for intratracheal instillation of materials to the entire lung in rats. Journal of Toxicologic Pathology. 19, (2), 107-109 (2006).
  26. Watanabe, A., Hashimoto, Y., Ochiai, E., Sato, A., Kamei, K. A simple method for confirming correct endotracheal intubation in mice. Laboratory Animals. 43, (4), 399-401 (2009).
  27. Kim, J. -S., et al. An automatic video instillator for intratracheal instillation in the rat. Laboratory Animals. 44, (1), 20-24 (2010).
  28. Lawrenz, M. B., Fodah, R. A., Gutierrez, M. G., Warawa, J. Intubation-mediated intratracheal (IMIT) instillation: a noninvasive, lung-specific delivery system. Journal of Visualized Experiments. (93), e52261 (2014).
  29. Ordodi, V. L., Mic, F. A., Mic, A. A., Sandesc, D., Paunescu, V. A simple device for intubation of rats. Lab Animal. 34, (8), 37-39 (2005).
  30. Cleary, G. M., Wiswell, T. E. Meconium-stained amniotic fluid and the meconium aspiration syndrome. An update. Pediatric Clinics of North America. 45, (3), 511-529 (1998).
  31. Vandivort, T. C., An, D., Parks, W. C. An improved method for rapid intubation of the trachea in mice. Journal of Visualized Experiments. (108), e53771 (2016).
  32. Litvin, D. G., Dick, T. E., Smith, C. B., Jacono, F. J. Lung-injury depresses glutamatergic synaptic transmission in the nucleus tractus solitarii via discrete age-dependent mechanisms in neonatal rats. Brain, Behavior, and Immunity. 70, 398-422 (2018).
  33. Spoelstra, E. N., et al. A novel and simple method for endotracheal intubation of mice. Laboratory Animals. 41, (1), 128-135 (2007).
  34. Kim, Y. E., et al. Intratracheal transplantation of mesenchymal stem cells simultaneously attenuates both lung and brain injuries in hyperoxic newborn rats. Pediatric Research. 80, (3), 415-424 (2016).
  35. Chang, Y. S., et al. Intratracheal transplantation of human umbilical cord blood-derived mesenchymal stem cells dose-dependently attenuate hyperoxia-induced lung injury in neonatal rats. Cell Transplantation. 20, (11-12), 1843-1854 (2011).
  36. Chang, Y. S., et al. Timing of umbilical cord blood derived mesenchymal stem cells transplantation determines therapeutic efficacy in the neonatal hyperoxic lung injury. PloS One. 8, (1), 52419 (2013).
  37. Mowat, V., Alexander, D. J., Pilling, A. M. A comparison of rodent and nonrodent laryngeal and tracheal bifurcation sensitivities in inhalation toxicity studies and their relevance for human exposure. Toxicologic Pathology. 45, (1), 216-222 (2017).
A Minimally Invasive Method for Intratracheal Instillation of Drugs in Neonatal Rodents to Treat Lung Disease
Play Video

Cite this Article

Sudhadevi, T., Ha, A. W., Harijith, A. A Minimally Invasive Method for Intratracheal Instillation of Drugs in Neonatal Rodents to Treat Lung Disease. J. Vis. Exp. (174), e61729, doi:10.3791/61729 (2021).More

Sudhadevi, T., Ha, A. W., Harijith, A. A Minimally Invasive Method for Intratracheal Instillation of Drugs in Neonatal Rodents to Treat Lung Disease. J. Vis. Exp. (174), e61729, doi:10.3791/61729 (2021).

Copy Citation Download Citation Reprints and Permissions
View Video

Get cutting-edge science videos from JoVE sent straight to your inbox every month.

Waiting X
Simple Hit Counter