Stem Cell CNTF Promotes Olfactory Epithelial Neuroregeneration and Functional Recovery Following Injury

Neurology Basic Medical Sciences Cell Biology Biochemistry Life Sciences Medicine
olfactory epithelium stem cells neuroregeneration CNTF inflammation functional recovery

Research Background and Academic Significance

Olfaction is one of the most important ways for humans to sense the external environment, fundamentally relying on olfactory sensory neurons (OSNs) in the olfactory epithelium (OE). These neurons possess the ability to regenerate throughout life, primarily due to the presence of local basal stem cell populations—namely, horizontal basal cells (HBCs) and globose basal cells (GBCs). Under normal physiological conditions, GBCs are mainly responsible for dividing and differentiating into new OSNs, while HBCs remain quiescent and are only activated after large-scale OSN injury to help replenish or repair tissue structure.

Following acute inflammatory damage from chemical or viral infections (such as COVID-19), although OSNs are rapidly lost (resulting in hyposmia or anosmia), the stem cell pool is generally spared, providing a possibility for spontaneous regeneration and functional recovery of the OE. However, clinical investigations and previous literature both indicate that a considerable proportion of patients cannot completely recover olfaction by themselves after severe olfactory injury, and there remains a lack of effective drugs or interventions targeting regenerative mechanisms. Identifying and elucidating the functions of key molecules within the stem cell–inflammation–regeneration axis is of great significance for understanding the basis of olfactory regeneration and developing new therapies.

This study focuses on Ciliary Neurotrophic Factor (CNTF)—a cytokine that mediates neuroprotection and neurogenesis after central nervous system injury. Previous work from the authors’ team found that HBCs can highly express CNTF, while GBCs express its receptor CNTF receptor α (CNTFRα), suggesting this signaling axis might regulate GBC proliferation and differentiation after injury. This research systematically aims to answer the following core scientific questions:

  1. Is CNTF expression in HBCs upregulated after acute inflammatory injury to the OE?
  2. What role does CNTF play in GBC proliferation and new OSN generation?
  3. Does CNTF signaling directly influence olfactory behavior and functional recovery?
  4. Can these mechanisms provide new pharmacological targets for functional restoration after olfactory injury?

Article Source and Author Information

The article, titled “stem cell cntf promotes olfactory epithelial neuroregeneration and functional recovery following injury,” is a collaborative work by Derek Cox, Brian Wang, Joe Oliver, Jaeden Pyburn, Diego J. Rodriguez-Gil, Theo Hagg, Cuihong Jia, et al., primarily affiliated with the Department of Biomedical Sciences, Quillen College of Medicine, East Tennessee State University, USA. The paper was published in 2025 in the renowned Oxford University Press journal “Stem Cells” (DOI: 10.1093/stmcls/sxaf033) and is available as open access.

Research Workflow and Technical Approach

1. Experimental Animals and Model Establishment

A total of 312 mice with various genotypes were used in this study, including CNTF knockout (cntf-/-) and wild-type (cntf+/+) littermate controls, C57BL/6 mice, and CK5Cre-Tdtomato transgenic mice for cell tracing. The CNTF knockout line was backcrossed for nine generations (99.8% C57BL/6 background) to ensure genetic consistency.

Acute olfactory epithelial inflammatory injury was induced by intraperitoneal injection of methimazole (MMZ, 75 mg/kg). This compound specifically destroys OSNs and triggers a local immune response. Some experiments included concurrent injection of bromodeoxyuridine (BrdU) to label proliferating cells, enabling cellular localization and functional analysis.

2. Expression Analysis of CNTF and Related Molecules

The authors quantitatively analyzed the expression of CNTF, LIF (Leukemia Inhibitory Factor), inflammatory factors (TNFα, IL-6, CD45), and GBC and stem cell proliferation markers (MASH1, KI67, PCNA, SOX2) in OE tissues at different time points via real-time quantitative PCR (RT-qPCR) and Western blot. In some experiments, primary HBCs were isolated and cultured to measure their spontaneous and injury-induced secretion and regulation of CNTF.

3. Dynamic Analysis of OE Stem Cell Proliferation and Differentiation

Immunohistochemical methods and BrdU in vivo pulse-chase techniques were used to perform spatiotemporal quantitative localization analysis of the proliferation dynamics of HBCs and GBCs (labeled by CK5 and MASH1, respectively), as well as the regenerative capacity of OSNs (OMP marker). Three-dimensional quantification was performed using confocal fluorescence microscopy to clarify stem cell fate in various genotypes.

4. Olfactory Functional Behavioral Assessment

Two classic mouse behavioral tests were used: the Buried Food Test (reflecting novel odorant detection and localization ability) and the Olfactory Habituation/Dishabituation Test (examining the ability to recognize and distinguish old versus new odors). All behavioral tests were conducted fully blind to exclude subjective bias from operators and analysts.

5. Data Statistics and Analysis

After data collection, groups were analyzed using two-sample t-tests, one/two-way ANOVA with Bonferroni post hoc tests, or two-way repeated measures ANOVA, with GraphPad Prism software. Results are presented as mean ± SD, with p<0.05 considered statistically significant.

Detailed Major Experimental Results

1. Acute Injury Induces High CNTF Expression in HBCs

Three to five days after methimazole-induced acute injury of the OE, CNTF mRNA and protein expression levels in tissue increased by 2–5 fold. Correlated inflammatory molecules (TNFα, IL-6, CD45) were also elevated. Primary cultured HBCs from the injury group showed significantly increased CNTF expression and secreted protein compared to controls, with no increase in inflammatory factor expression, suggesting HBCs are the main post-inflammation CNTF source rather than a source of inflammatory factors or LIF. Injury-group HBCs also showed stronger expression of proliferation markers (KI67, PCNA).

2. Key Role of CNTF Signaling in GBC Proliferation

Analysis of GBC differentiation and proliferation in CNTF+/+ and CNTF-/- mice before and after injury showed significantly higher mRNA levels of GBCs (MASH1+ cells) in the CNTF+/+ group than in CNTF-/- after methimazole treatment. BrdU tracing revealed a significant increase in BrdU+ cells (over 96% were GBCs) in the wild-type injury group compared to controls, while CNTF-/- mice had only a limited, non-significant proliferation increase, with a 33%-40% deficit compared to wild-type. Cell co-labeling indicated that approximately 80% of proliferating cells after injury were MASH1+ GBCs, only 1.5% were CK5+ HBCs, and inflammatory cells (CD45+) were excluded. This demonstrates that injury activates HBC-CNTF secretion, which then specifically regulates GBC proliferation.

3. CNTF Deficiency Impairs Neuroregeneration but Not GBC Self-renewal

With BrdU pulse-chase tracking, dynamic regeneration of OSNs and GBCs after injury at different time points was compared between CNTF+/+ and CNTF-/- mice. At three weeks post-injury, the CNTF+/+ group had more BrdU+ cells in the middle layer (area of new OSN differentiation) compared to CNTF-/-, but there was no statistical difference between groups in the basal layer (GBC self-renewal). Six-week follow-ups showed BrdU+ cell numbers returned to baseline because of the natural OSN apoptosis in both groups, with no significant difference, suggesting that CNTF mainly promotes GBC→OSN differentiation and has limited impact on GBC self-renewal.

4. CNTF Deficiency Leads to Delayed Olfactory Functional Recovery

Behavioral assessments showed that all mice failed to find buried food three days after injury, and the habituation/dishabituation test also showed no olfactory discrimination. At three weeks, CNTF+/+ mice displayed significant functional recovery in both behavioral assessments, while CNTF-/- mice showed little to no recovery. At six weeks, CNTF+/+ mice fully recovered to control levels, but CNTF-/- still exhibited marked delays. These changes could not be attributed to motor or hunger motivation differences, further underscoring the physiological importance of CNTF.

Research Conclusions and Scientific Value

This study systematically reveals the core mechanisms of the HBC-CNTF-GBC signaling axis following OE injury: after acute injury, HBCs are activated to highly express and secrete CNTF, which acts in a paracrine fashion to promote GBC proliferation and differentiation into new OSNs, thereby driving neuroregeneration and olfactory function recovery. CNTF deficiency severely impairs GBC regeneration, OSN formation, and leads to delayed or incomplete functional recovery of olfaction.

These findings not only deepen our molecular-level understanding of the olfactory epithelial regenerative microenvironment and provide a solid theoretical foundation for the mechanisms underlying spontaneous olfactory functional repair but also offer new perspectives for future drug development targeting CNTF, especially for interventions against acute inflammatory or virus-related (such as COVID-19) olfactory disorders.

Research Highlights and Innovations

  1. For the first time, the nodal roles of HBCs and CNTF in the olfactory epithelial neuroregeneration chain were clearly defined at the molecular and cellular level, elucidating the key limiting factors in regeneration following acute inflammation-induced injury.
  2. The study combined animal models, cell culture, and multidimensional behavioral verification to establish a complete causal chain and enhance the reliability of the conclusions.
  3. It distinguished the effects of CNTF on GBC differentiation versus self-renewal in detail, supporting stem cell biology theories.
  4. It provides a scientific basis for designing drug interventions for olfactory epithelial regeneration, especially the application prospects of local (intranasal) delivery of CNTF-like drugs.

Other Important Information

  • The article declared no potential conflicts of interest, and all data can be requested from the corresponding author.
  • The research team received rigorous support from several NIH grants (including R01DC020528, R01NS102745, etc.).
  • The references are comprehensive, thoroughly reviewing and building on the existing literature for olfactory epithelial stem cell regeneration and CNTF biology.

Summary and Outlook

This in-depth basic study not only enriches the academic landscape for stem cell-regulated olfactory epithelium regeneration, but also highlights the therapeutic potential of the CNTF axis in functional recovery after inflammation and injury. In the future, exploring CNTF gene delivery, nasal delivery of biological preparations, and other translational medical pathways may benefit patients with olfactory dysfunction and related neural regeneration deficits. This research will have long-term and profound implications for the field of neural repair, sensory disorder regenerative treatment, and stem cell therapeutic strategies.