Abstract
Background/Aim: Wound healing is difficult to study due to interspecies variation in healing mechanisms, rates, and genetic factors. This study aimed to characterize tracheal wound healing in Wistar rats (Rattus norvegicus) following a thin, partial-thickness injury using a modified Derby-Perry Excavator.
Materials and Methods: Four male Wistar rats underwent endotracheal wounding under ketamine anesthesia. Each rat was euthanized at one, three, five, or six days post-injury. Excised tracheas were bisected for histological examination (Hematoxylin and Eosin staining) and quantitative RT-PCR analysis of COL3A1 mRNA expression.
Results: Partial-thickness tracheal injury resulted in accelerated wound healing. Histology and COL3A1 expression indicated rapid progression of healing phases. Inflammation resolved by day 3, with the proliferative phase beginning around day 2, peaking at day 3, and transitioning to maturation by day 5. The rapid timeline likely reflects the small, superficial nature of the wound.
Conclusion: Partial-thickness tracheal injury using a modified Derby-Perry Excavator produced a reproducible, rapidly healing model suitable for studying airway repair mechanisms.
Introduction
Wound healing is a complex process that occurs after injury. The wound healing process encompasses four overlapping phases, i.e., hemostasis, inflammation, proliferative, and remodeling phases. Hemostasis normally occurs soon after injury, allowing bleeding to stop through platelet and erythrocyte adherence to disrupted endothelium, activation of platelet and the release of platelet alpha granules (1). The following inflammation phase ensues. Several cells play a huge role in this phase, i.e., neutrophils and macrophages, with the primary goal of removing infectious agents, debris, and necrotic tissue. Once the wound has been debrided, macrophage becomes the M2 subset and release growth factors, such as transforming growth factor β (TGFβ), platelet derived growth factor (PDGF), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF), which are crucial factors for the proliferative phase. The proliferative phase begins to produce granulation tissue and induce re-epithelialization. The subsequent remodeling phase completes the wound healing process. It is worth noting that these phases may overlap (1–5).
The wound healing process is influenced by several factors, one of which is lesion size (4). A study by Grendel et al. described the wound healing phases in the trachea of Sprague-Dawley rats after tracheostomy (6). At 7 days, polymorphonuclear leukocytes presented with well-formed granulation tissue. At 14 days, the number of macrophages increased; however, the number of polymorphonuclear leukocytes remained unchanged. The granulation tissue area began to decrease, while its collagen and angiogenesis content increased, indicating a transition from the proliferative to the remodeling phase. At 28 days, the granulation tissue area and angiogenesis continued to decrease, marking the middle of the remodeling phase. It can be inferred from this study that while the inflammation phase lasted for more than 14 days, the proliferative phase peaked around days 7-14, and the remodeling phase began from day 14 (6). As mentioned previously, the geometric size of a wound may have an impact on its healing rate. It cannot be assumed that a partial thickness and thin wound have the same wound healing time as a larger wound (4).
To our knowledge, the existing literature describes wound healing in tracheal rats in larger wound settings. Still, none have described the wound healing trends of smaller, more superficial wounds (6). In the effort of identifying articles on superficial endotracheal wounds, the combination of MeSH keywords: (“Superficial Wound” OR “Partial Thickness Wound”) AND (Healing Time OR Healing Rate) AND (trachea) AND (rats OR “wistar rats” OR “wistar rat” OR “rattus norvegicus”) were employed in four databases - PubMed, EuroPNC, EBSCO and Scidirect. The search results yielded 0 results in PubMed, 31 in EuroPMC, 0 in EBSCO and 73 in Scidirect. After initial screening of titles, none were deemed relevant to our inquiry.
This study, therefore, aimed to describe the wound healing process in the trachea of Wistar rats (Rattus norvegicus) after tracheal wounding using a modified Derby-Perry excavator, in which the partial-thickness wound made by the excavator was thin. The wound healing process was evaluated using two methods, i.e., histological examination with Hematoxylin and Eosin (H&E) staining and quantitative reverse transcription polymerase chain reaction (qRT-PCR) of COL3A1 mRNA.
Materials and Methods
Animal husbandry and treatment. All animal experimental protocols were conducted in the Animal Facility of Animal Research and Innovation Center, Padjadjaran University, Jatinangor in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals. This study protocol was approved by the Padjadjaran University Ethics Committee (#759/UN6.C.6.30/PT.00/2024). A total of four male Wistar rats, 12 weeks old and weighting 230-241 g, were purchased from the School of Life Sciences and Technology, Bandung Institute of Technology. All animals were housed in polycarbonate plastic cages (four rats per cage) with wood-shaving bedding, maintained in a controlled room at temperature 25±2°C, with humidity 50±10%, and 12/12 h of light-dark cycles with ad libitum access to food and water (8).
Endotracheal wounding protocol. The endotracheal wounding was performed based on Hillel et al. with modification (7). The rat was positioned in a supine posture on a specialized rat intubation table (patented by JI, patent number EC002025045045), with its incisors secured using a rubber band to maintain head stability. A pen light equipped with an acrylic attachment was used to visualize key anatomical structures, including the glottis, epiglottis, and trachea. Following clear visualization, a modified Derby Perry dental excavator (Bertamed, Indonesia) was carefully inserted into the trachea, and a controlled swiping motion was performed three times to induce a standardized tracheal injury. After the procedure, the rat was immediately turned to a prone position to minimize the risk of airway obstruction and aspiration.
Trachea excision protocol. The rats were sacrificed at different times, i.e., one day, three days, five days, and six days post-wounding, using intravenous administration of ketamine (PT. Ethica Industry Farmasi, Indonesia), without performing cervical dislocation. Following euthanasia, the skin and subcutaneous tissue were excised to expose the underlying structures. The sternomastoid muscle was removed to allow for clear visualization of the trachea. The trachea was carefully excised at two anatomical landmarks, i.e., the thyroid cartilage and just above the carina. After excision, the trachea was measured and divided into two equal segments. The proximal portion was placed in a microtube containing formaldehyde and sent to the Histology Laboratory at Hasan Sadikin General Hospital in Bandung. Meanwhile, the distal portion was stored in a microtube, placed in a receptacle filled with liquid nitrogen, and transported to the Genomics Laboratory at the Central Laboratory, Padjadjaran University, Jatinangor for qRT-PCR to detect COL3A1 mRNA.
Histological examination. The proximal trachea was embedded into paraffin blocks and transverse cuts were made. Following that process, the tissue was stained with Haematoxylin Mayer and Eosin Floxin 1%. Histology slides were examined and interpreted by an anatomical pathologist.
Quantification of COL3A1 mRNA expression. RNA extraction was performed using GENEzol Reagent (Geneaid, New Taipei, Taiwan, ROC) to obtain a pellet, to which 200 μl of 70% ethanol was added. The mixture was centrifuged for 5 min at 11,200 RPM at 4°C. The resulting pellet was air-dried for 5–10 min at room temperature, after which 20 μl of nuclease-free water (NFW) (HiMedia Laboratories, Maharashtra, India) was added. The solution was subsequently incubated in a heat block at 60°C for 10 min to ensure complete dissolution of RNA. RNA concentration was then quantified using a NanoQuant Plate Tecan Multimode reader to confirm sufficient RNA yield. A master mix solution was prepared by combining SYBR green dye, forward and reverse COL3A1 primers, RNase inhibitor, reverse transcriptase enzyme, NFW, and the RNA template obtained from the earlier steps. The prepared master mix was aliquoted into microtube wells and processed using the Agilent Technologies ARIAMX Real-Time PCR System (Agilent Technologies, Singapore). To determine the fold change in COL3A1 mRNA expression, the Livak method (2−ΔΔCt) was used, with β-actin serving as the housekeeping gene for normalization (8). The expression of COL3A1 gene results in type III collagen. During wound healing, the ratio of type III to type I collagen changes. An increase in COL3A1 mRNA expression indicates the proliferative phase, while its decrease suggests the maturation phase (1, 4).
Results
Rat A was sacrificed one day after wounding, Rat B was sacrificed at three days after wounding, Rat C was sacrificed five days after wounding, and Rat D was sacrificed six days after wounding. Prior to sacrifice, each rat was evaluated daily after wounding. Two primary outcomes were assessed: weight (in grams) and presence of wheezing. Table I and Figure 1 depict weight trends of each rat. None of the rats experienced wheezing or natural death. Table II and Figure 2 summarize the histologic findings of the wounded trachea one day, three days, five days, and six days after wounding. Fold change of COL3A1 mRNA expression per rat is depicted in Table III and Figure 3.
Daily weight progression and survival timeline of rats following tracheal wounding.
Weight trends of each rat. The dark blue line represents weight trend of Rat A, orange line represents weight trend of Rat B, green line represents weight trend of Rat C and light blue line represents weight trend of Rat D.
Histologic findings and COL3A1 mRNA fold-change measurements at each sacrifice time point.
Histologic appearance of a wounded rat trachea. A) At 4′ magnification, epithelial disruption appeared. Loose connective tissues appear edematous. B) At 10×magnification, epithelial disruption along with chondrocyte disruption are noted (arrow). Glands that were originally from outer regions invaginated towards the tracheal wall (*). C) Epithelial disruption and PMNs in the lumen are noted (arrows). Lymphocytes are seen in the epithelial tissue. Intravascular monocytes undergoing diapedesis are also noted (arrowhead). Connective tissue appeared edematous (*). D) At 4×magnification, epithelial disruption is present in ¼ regions. The presence of incomplete reepithelization is observed in ¾ of the regions. E) At 10×magnification, lymphocytes are seen in the lumen. F) At 4 ×magnification, re-epithelialization occurs in all regions. G) At 4 ×magnification, re-epithelialization occurs in all regions, accompanied by the presence of cilia.
CT value, ΔCT and ΔΔCT values and fold-change quantification using Livak’s method.
Fold-change values of COL3A1 mRNA expression in Rats A–D. Rat A, B, C, and D had a fold change value of 0.73, 1.00, 0.19, and 0.38, respectively.
Through histologic examination, epithelial disruption was observed on day 1. While mononuclear cells and eosinophils were present, polymorphonuclear cells were present only in the lumen (Figure 2A-C). In addition, the fold change value of COL3A1 mRNA obtained was 0.73. Histologic examination on day 3 showed re-epithelialization had occurred in 75% parts of the trachea. Only a few mononuclear inflammatory cells were present in the lumen (Figure 2D and E). An increment of fold change value was observed as the fold change value of COL3A1 mRNA was 1. On day 5, re-epithelialization had occurred in all parts. No inflammatory cells were observed (Figure 2F). A decrease in fold change value was observed: the fold change value of COL3A1 mRNA obtained was 0.19. Re-epithelialization had occurred in all parts with the presence of cilia on day 6 (Figure 2G). An increment in the fold change value of COL3A1 mRNA to 0.38 was observed. Histologic findings indicate that using this wounding method, wound healing completes at 6 days.
The COL3A1 mRNA fold change correlated with wound healing phases. Rat A (rat sacrificed at 1 day post-wounding) depicted the inflammatory phase nearing completion, as mononuclear cells had begun to appear on this day (5). As the inflammatory and proliferative phases may co-occur, this could explain the presence of COL3A1 mRNA (1, 3). A higher fold change was seen in Rat B (rat sacrificed at 3 days post wounding). Histologically, Rat B was undergoing the proliferative phase as explained by the fewer inflammatory cells observed, and 75% of the tracheal epithelium had undergone early re-epithelialization. Rat C (rat sacrificed at 5 days post-wounding) depicted that re-epithelialization had commenced in all regions. The decrease in COL3A1 mRNA fold change describes the end of the proliferative phase and the start of the maturation phase. Rat D demonstrated the completion of re-epithelialization with cilia in all regions present.
Figure 4 describes the wound healing phases proposed based on the histological and COL3A1 mRNA fold change findings. It can be inferred from both aspects that tracheal wound healing occurs sooner than what was described by Grendel et al. (Table IV) (6).
Wound healing phases proposed from this research.
Comparison of wound-healing progression between the present study and Grendel et al. (6).
Discussion
Injuries are classified into two types: mechanical and burn injuries. The healing time and formation of granulation tissue of each wounding technique differ from one another. A thermal burn model using Sprague-Dawley rats reported that inflammatory cells were present seven days post-wounding, and the formation of granulation tissue occurred at 21 days after wounding (8).
Injury formed in our study was a mechanical injury. As mentioned previously, Grendel et al. described in their tracheostomy rat model, the inflammation phase lasted for more than 14 days, the proliferative phase peaked around days 7-14, and the remodeling phase began from day 14 (6). Our research findings differed from previous literature. Grendel et al. reported that PMNs were still present at seven days post-wounding, along with the presence of granulation tissue. At 14 days post-wounding, mononuclear (MN) cells began to dominate, and the granulation tissue began to decrease in size. At 28 days, it can be inferred that the maturation phase was still in progress. This research found that the timeline of wound healing was quicker. Table IV summarizes the difference in findings between this research and Grendel et al.’s.
Neither this study nor the one by Grendel et al. included any interventions –such as drug administration–that could influence the wound-healing process in the observed rats. Therefore, tracheal wound healing occurred sooner with this protocol due to several factors, including the depth and size of the wound. In this study, wounds were created using modified Derby Perry excavators, resulting in partial-thickness, shallow tracheal injuries. The smaller size and more superficial depth explained the faster wound healing process that occurred in our research. A study by Gorin et al. evaluated the impact of initial wound geometry (size and depth) and its wound healing rate. Two methods were used to measure wound healing rate: calculating the linear healing rate using Gilman’s Equation and assessing the change in wound area per day. The change in wound area per day was influenced by initial wound geometrics including area, perimeter, length and width. Regression analysis was performed and r=0.85 was obtained, indicating strong correlation between change in wound area per day and initial wound geometrics (9).
Interestingly, COL3A1 mRNA expression increased in Rat D compared with Rat C. Theoretically, COL3A1 mRNA levels should continue to decline during the maturation phase, as this stage is characterized by a decreasing type III:type I collagen ratio due to the gradual replacement of type III collagen by type I collagen (3, 10, 11). Several factors can cause abnormality in the replacement of type 3 collagen to type 1 collagen. These factors include prolonged inflammation or dysregulation of TGFβ growth factor. Although inflammatory cells were not observed in rat D, endotracheal wound evaluation and workup of serum inflammatory markers were not performed. Therefore, the presence of previous prolonged inflammation cannot be ruled out and may be a possible etiology of this abnormal finding.
Conclusion
Partial-thickness tracheal injury resulted in accelerated wound healing. Histology and COL3A1 expression indicated rapid progression of healing phases. Inflammation resolved by day 3, with the proliferative phase beginning around day 2, peaking at day 3, and transitioning to maturation by day 5. The rapid timeline likely reflects the small, superficial nature of the wound.
Acknowledgements
The Authors would like to express their warmest gratitude to the teams of Lab Sentral Padjadjaran University, Animal Research Innovation Center Padjadjaran University, and Histology Lab of Hasan Sadikin General Hospital for making this study possible.
Footnotes
Authors’ Contributions
J.I. was the principal investigator and in charge of the conception of the manuscript. J.I., A.S.K., I.I., A.K. and J.J. supervised the study. A.Y. performed an examination of histology slides. R.L., A.M.R. and S.A.R. conducted the study. All Authors prepared the manuscript and agreed to submit the final version of this manuscript.
Conflicts of Interest
The Authors declare that they have no known conflicts of interest that could have influenced the work reported in this paper.
Funding
The present study was funded by Universitas Padjadjaran through the Indonesian Endowment Fund for Education (LPDP) on behalf of the Indonesian Ministry of Higher Education, Science and Technology and managed under the EQUITY Program (Contract No. 4303/B3/DT.03.08/2025 and 3927/UN6.RKT/HK.07.00/2025).
Artificial Intelligence (AI) Disclosure
During the preparation of this manuscript, a large language model (LLM) was used solely for language editing and stylistic improvements in select paragraphs. No sections involving the generation, analysis, or interpretation of research data were produced by generative AI. All scientific content was created and verified by the authors. Furthermore, no figures or visual data were generated or modified using generative AI or machine learning–based image enhancement tools.
- Received November 10, 2025.
- Revision received November 18, 2025.
- Accepted November 19, 2025.
- Copyright © 2026 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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