Effects of GlyNAC on Tissue Regeneration, Behavior, and Locomotor Activity in Dugesia tigrina

== Abstract ==
This study investigates the effects of GlyNAC—a combination of glycine and N-acetylcysteine (NAC)—on planarian tissue regeneration, locomotor behavior, and eyespot reformation. As organisms age, tissue regeneration declines, partly due to a deficiency of glutathione (GSH), a key antioxidant synthesized from glycine and cysteine. GlyNAC has been shown to restore GSH levels and reverse aging-associated deficits in humans; however, its effect on in vivo tissue regeneration in a whole-organism model has not been evaluated. Dugesia tigrina planarians were used as the model organism due to their pluripotent neoblast-driven regeneration, similar central nervous system organization to vertebrates, and endogenous GSH redox system. Forty planarians were assigned to four groups (n = 10 per group) receiving ground beef mixed with 0%, 1%, 5%, or 10% GlyNAC (g/g) for seven days prior to transverse amputation. Following amputation, eyespot regeneration and fragment body length were recorded every Monday, Wednesday, and Friday for 11 days. Locomotor activity was assessed by counting grid-line crossings during 30-minute video recordings before and after treatment. Statistical analysis used one-way analysis of variance (ANOVA) followed by Tukey's honest significant difference (HSD) post hoc test (significance threshold: α = 0.05). Results indicated that GlyNAC increased eyespot regeneration and fragment length relative to controls, supporting the hypothesis that GlyNAC promotes tissue regeneration. Limitations, including manual movement scoring, scheduling gaps, and planarian mortality at 10% GlyNAC in Trial 1 are discussed. Future research should employ automated tracking, longer treatment durations, and larger sample sizes.

Keywords: Dugesia tigrina, GlyNAC, glutathione, tissue regeneration, oxidative stress, locomotor activity

== 1. Introduction ==

=== 1.1 GlyNAC and Aging ===
There has been growing interest in anti-aging research due to the widespread symptoms associated with aging, including muscle fatigue, chronic inflammation, decreased tissue regeneration, and oxidative damage [1]. Oxidative stress occurs when there is an imbalance between the production of reactive oxygen species (ROS) and the body's ability to detoxify them [2]. Excessive oxidative stress accelerates aging and cell death, whereas low oxidative stress supports cell regeneration [3].

Despite limited mechanistic understanding of why older individuals experience these symptoms, many attempt to preserve health through dietary strategies, supplements, and antioxidants. Previous research has demonstrated that both glycine and N-acetylcysteine (NAC) independently protect against oxidative stress [4,5]. Crucially, these compounds serve as direct precursors to glutathione (GSH), a master intracellular antioxidant that defends against oxidative damage, supports energy metabolism, and promotes cell proliferation [6]. GlyNAC—a combined supplement of glycine and NAC—has been shown to restore GSH levels and reverse multiple aging-associated health deficits in older adults [1].

With aging, GSH deficiency arises from insufficient availability of glycine and cysteine required for GSH synthesis. The resulting decline in antioxidant defenses allows oxidative stress to accumulate, impairing cells that sustain tissue homeostasis. GlyNAC supplementation thus represents a rational strategy for restoring antioxidant capacity and supporting regenerative function in aging organisms.

=== 1.2 Dugesia tigrina as a Model Organism ===
Planarians, specifically Dugesia tigrina, are freshwater flatworms renowned for their exceptional tissue regeneration capacity. When bisected, each fragment activates a population of pluripotent adult stem cells called neoblasts, which proliferate and differentiate to reconstitute the entire organism within days [7]. This process relies on stem cell mechanisms that parallel vertebrate tissue repair, making planarians a broadly informative model for regenerative biology.

Planarians also possess an endogenous GSH-based redox system that is essential for balancing oxidative status during regeneration [8]. This makes D. tigrina particularly well-suited to investigate whether GlyNAC-driven GSH augmentation can enhance regenerative outcomes. Additionally, the species exhibits quantifiable behavioral outputs—including locomotor activity and stereotyped aversive responses—that provide indirect readouts of nervous system integrity and overall physiological condition.

The present study was designed to test whether dietary GlyNAC supplementation at graded concentrations (0–10% g/g in ground beef) would enhance tissue regeneration, eyespot reformation, and locomotor activity in D. tigrina. A positive result would support further investigation of GlyNAC in vertebrate regeneration models and strengthen the translational case for its use in aging populations.

== 2. Materials and Methods ==

=== 2.1 Preparation of GlyNAC Treatment ===
Glycine and N-acetylcysteine were purchased from a commercial supplier (Nutricost, via Amazon). The two compounds were combined in a 1:1 mass ratio to produce GlyNAC, which was then blended into ground beef at target concentrations of 0%, 1%, 5%, and 10% (g/g) for groups A–D, respectively (Table 1). In Trial 2, the Group D concentration was reduced from 10% to 5% following substantial planarian mortality observed at the 10% concentration in Trial 1.

==== Table 1. Composition of GlyNAC treatment diets by group. ====
{|
|Group
|GlyNAC in Beef (% g/g)
|GlyNAC (g)
|Glycine (g)
|NAC (g)
|Ground Beef (g)
|n
|-
|A
|0
|0
|0
|0
|20
|10
|-
|B
|1%
|0.2
|0.1
|0.1
|19.8
|10
|-
|C
|5%
|1.0
|0.5
|0.5
|19
|10
|-
|D*
|10% (T1); 5% (T2)
|2.0 / 1.0
|1.0 / 0.5
|1.0 / 0.5
|18 / 19
|10
|}
<nowiki>*</nowiki>Group D concentration was reduced to 5% in Trial 2 following mortality observed at 10% in Trial 1.

=== 2.2 Organism Husbandry ===
Forty Dugesia tigrina planarians were obtained from a commercial supplier (Carolina Biological Supply, catalog no. 132954) and randomly assigned to four groups of ten. Animals were housed individually in loosely sealed glass jars containing Poland Spring water and stored in a darkened cabinet at room temperature. Planarians were acclimated for seven days on plain ground beef (three feedings per week: Monday, Wednesday, Friday) prior to experimental treatment. Following each feeding, leftover beef was removed and fouled water was replaced with fresh Poland Spring water using a pipette, taking care not to aspirate any organisms. All handling was performed with appropriate personal protective equipment (PPE), and work surfaces were disinfected with a 10% bleach solution before each session.

=== 2.3 Treatment Administration ===
Following acclimation, each group received 20 g of the appropriate GlyNAC-supplemented (or control) ground beef preparation up to three times per week for seven days prior to amputation. Water was replaced and residual food was removed after each feeding as described in Section 2.2.

2.4 Locomotor Activity Assessment

Baseline locomotor activity was recorded before treatment initiation. For each session, 30 mL of Poland Spring water was added to four sterile Petri dishes placed over graph paper grids (Fig. 1). Planarians from each group were transferred to their respective dishes, allowed a brief acclimation period, and then video-recorded for 30 minutes. The number of grid lines crossed by each individual was counted from the recordings as a proxy for locomotion. This procedure was repeated after the treatment period to assess any treatment-related changes in motility.
[[File:Screenshot 2026-05-30 at 12.27.43 PM.png|centre|thumb]]

==== Fig. 1. Experimental setup for locomotor activity assessment. Petri dishes placed over graph paper grids to enable quantification of grid-line crossings as a locomotion proxy.Planarian Cutting ====

==== 2.5 Transverse Amputation ====
Prior to amputation, planarians were cold-anesthetized on ice for one minute. Each animal was transversely bisected anterior to the pharynx using a scalpel sterilized with 70% ethanol; the scalpel was re-sterilized after every three cuts to prevent cross-contamination. Head and tail fragments from each group were placed into separately labeled containers (e.g., AH = Group A heads; AT = Group A tails) containing the appropriate treatment solution. The average mass of each fragment group (AH, BH, CH, DH, AT, BT, CT, DT) was recorded immediately following amputation.

=== 2.6 Regeneration Assays ===
Fragment body length (mm) and eyespot regeneration were assessed every Monday, Wednesday, and Friday for 11 days following amputation. Eyespot appearance in tail fragments was observed under a dissecting microscope using a three-point ordinal scale: 0 = absent; 0.5 = partial; 1 = fully formed bilaterally symmetric eyespots. Fragment lengths were measured with a ruler and recorded in Microsoft Excel. After the completion of both trials, all planarians were submitted to a faculty supervisor for ethical euthanasia by autoclaving at 121°C.

=== 2.7 Statistical Analysis ===
<nowiki>Group means and standard deviations were calculated using standard Excel functions. Between-group differences were assessed by one-way ANOVA followed by Tukey's HSD post hoc test using an online statistics platform (https://www.socscistatistics.com/tests/anova/default2.aspx). A significance threshold of α = 0.05 was applied. Locomotor data were graphed as scatter plots of grid-line crossings per day; regeneration data were presented as bar graphs of growth from Day 0 to Day 11 and line graphs of mean eyespot scores over time.</nowiki>

== Result ==

[[File:Screenshot 2026-06-14 at 1.52.37 PM.png|centre|thumb|Figure 2. Eyespot regeneration over time in Trial 1 across four GlyNAC concentrations (0%, 1%, 5%, 10%). n=10 per group.]]To evaluate the effect of GlyNAC on cranial tissue repair, eyespot regeneration was monitored over 5 days in Trial 1 across four concentrations (0%, 1%, 5%, and 10%), with n = 10 planarians per group. As shown in Figure 2, the 0% control group exhibited the slowest regenerative progression, with eyespots remaining incompletely formed at the 5-day endpoint. The 1% and 5% GlyNAC groups demonstrated accelerated regeneration compared to controls, achieving complete eyespot formation approximately 3 days earlier than the control group. The 5% group showed the most rapid regeneration overall, with full eyespot restoration observed by day 4. The 10% group displayed intermediate recovery, with regeneration rates falling between the 1% and 5% groups.[[File:Screenshot 2026-06-14 at 1.58.45 PM.png|centre|thumb|Figure 3. Tail regeneration in Trial 1. Difference in tail length from day 1 to day 11. Error bars = standard deviation. *p < 0.05, *''p < 0.01'']]To evaluate the effect of GlyNAC on posterior tissue regrowth, the change in tail length from day 1 to day 11 was measured in Trial 1, with results presented in Figure 3. The 0% control group showed the smallest increase in tail length over the 11-day period. The 1% and 5% GlyNAC groups exhibited greater increases in tail length compared to controls, with increases ranging from 1.3 to 1.5 mm. The 5% GlyNAC group produced the largest increase in tail length among all groups, with a statistically significant difference from the control (*p* < 0.01). The 10% GlyNAC group also showed increased tail length relative to controls, though this increase was less pronounced than that observed in the 5% group. Error bars in Figure 3 represent standard deviation for each group.
[[File:Screenshot 2026-06-14 at 2.00.17 PM.png|alt=|centre|thumb|Figure 4. Head regeneration in Trial 1. Difference in head length from day 1 to day 11. Error bars = standard deviation]]As presented in Figure 4, head length increased across all groups from day 1 to day 11. The 0% control group showed the smallest increase in head length. The 1% and 5% GlyNAC groups demonstrated greater increases in head length compared to controls, with increases ranging from 1.3 to 1.5 mm. The 5% group exhibited the greatest increase in head length among all treatment groups. The 10% group showed increases between the 1% and 5% groups. Statistical analysis revealed that most comparisons between treatment groups and controls did not reach statistical significance.

[[File:Screenshot 2026-06-14 at 2.02.31 PM.png|centre|thumb|''Figure 5. Planarian movement in Trial 1. Gridlines crossed per planarian over time.'']]Planarian movement was quantified by counting grid-line crossings during 30-minute video recordings, with results shown in Figure 5. The 0% control group displayed baseline locomotor activity. The 1% and 5% GlyNAC groups showed increased grid-line crossings compared to controls, while the 10% group exhibited reduced activity relative to the 5% group. However, statistical analysis revealed no significant differences in locomotor activity across treatment groups.[[File:Screenshot 2026-06-14 at 2.02.38 PM.png|centre|thumb|''Figure 6. Eyespot regeneration over time in Trial 2.'']]In Trial 2, eyespot regeneration was monitored over time across GlyNAC concentrations (0%, 1%, and 5%, with the 10% dose replaced by 5% due to toxicity in Trial 1), as shown in Figure 6. The 0% control group showed the slowest progression toward full eyespot formation. The 1% and 5% GlyNAC groups demonstrated accelerated regeneration compared to controls, consistent with the patterns observed in Trial 1. The 5% group exhibited the most rapid regeneration, achieving full eyespot formation earlier than all other groups.[[File:Screenshot 2026-06-14 at 2.02.47 PM.png|centre|thumb|''Figure 7. Head regeneration over time in Trial 2.'']]Head regeneration over time in Trial 2 is presented in Figure 7. The 0% control group showed the smallest increase in head length. The 1% and 5% groups demonstrated moderate to large increases in head length compared to controls. The 5% group exhibited the greatest increase in head length among all groups. Most between-group comparisons did not reach statistical significance.[[File:Screenshot 2026-06-14 at 2.02.53 PM.png|centre|thumb|''Figure 8. Tail regeneration over time in Trial 2.'']]As shown in Figure 8, tail regeneration in Trial 2 followed a similar pattern to Trial 1. The 0% control group showed the smallest increase in tail length. The 1% and 5% groups exhibited greater increases in tail length compared to controls. The 5% group demonstrated the largest increase in tail length among all treatment groups. Statistical analysis revealed that most comparisons between treatment groups and controls did not reach statistical significance.[[File:Screenshot 2026-06-14 at 2.03.00 PM.png|centre|thumb|''Figure 9. Planarian movement in Trial 2. Gridlines crossed per planarian over time.'']]Planarian movement in Trial 2 is shown in Figure 9. The 0% control group displayed baseline locomotor activity. The 1% and 5% GlyNAC groups showed grid-line crossings comparable to or slightly increased from controls. Statistical analysis revealed no significant differences in locomotor activity across treatment groups.

== 4. Discussion ==
In this study, GlyNAC supplementation at 5% and 1% accelerated eyespot regeneration by approximately 3 days compared to controls and increased head and tail length growth by 1.3–1.5 mm. The data indicate that GlyNAC supplementation increased eyespot regeneration rate and fragment body length relative to the control group, supporting the hypothesis that GlyNAC exerts a positive effect on tissue regeneration in ''D. tigrina''. These findings are consistent with previous studies in other model organisms, where GlyNAC has been shown to enhance wound healing and tissue repair through glutathione-mediated pathways. Given the mechanistic parallels between planarian neoblast-driven regeneration and vertebrate stem cell repair [7], these findings suggest GlyNAC may similarly promote tissue regeneration in humans, potentially through restoration of GSH levels and reduction of oxidative stress. However, direct extrapolation to human physiology requires further investigation in mammalian models.

Several methodological limitations may have affected the results. Locomotor activity was quantified by manual counting of grid-line crossings from video recordings, which is susceptible to inter-observer variability and fatigue-related errors. Shortening the recording duration from 30 minutes was adopted as a partial mitigation, but did not eliminate observer-related error. Scheduling constraints and holidays created gaps in the data collection timeline, complicating comparison across trials and reducing the ability to detect gradual trends. Trial duration was insufficient to capture the full temporal extent of GlyNAC's effects. In Trial 1, all planarians in Group D (10% GlyNAC) died, indicating that this concentration is toxic to ''D. tigrina''; the concentration was reduced to 5% in Trial 2, which resolved the mortality issue and yielded informative data. Future studies should include a broader range of non-lethal concentrations (e.g., 0.5%, 2%, 5%) to identify the optimal therapeutic window. Additionally, automated tracking software should be considered for more objective and precise measurement of locomotor activity.

Despite these limitations, this study provides preliminary in vivo evidence that dietary GlyNAC supplementation can enhance planarian tissue regeneration. The finding that GlyNAC is most likely to benefit aging organisms due to their pronounced GSH deficiency has direct relevance to translational aging research. Future research should explore the dose-dependent effects of GlyNAC across multiple regeneration cycles and investigate the underlying molecular mechanisms, including neoblast proliferation rates and gene expression changes in antioxidant pathways. Replication of these findings in vertebrate models, such as zebrafish or murine wound-healing assays, would further strengthen the translational potential of GlyNAC for human regenerative medicine.

== 5. Conclusions ==
This study investigated whether dietary GlyNAC supplementation at graded concentrations (0–10% g/g) enhances tissue regeneration, eyespot reformation, and locomotor activity in Dugesia tigrina across two independent trials. GlyNAC-treated groups showed increased eyespot regeneration and fragment length relative to controls, consistent with the proposed mechanism of GSH restoration reducing oxidative barriers to neoblast activity. These results contribute to a growing evidence base for GlyNAC as a pro-regenerative intervention and support further investigation in vertebrate models.

Future work should employ automated tracking systems (e.g., thermal cameras or machine learning-based motion analysis) to reduce observer error in locomotor assessments. Alternative delivery routes, such as immersion in GlyNAC solution rather than dietary administration, should be evaluated to improve bioavailability. Extended treatment durations and larger sample sizes would strengthen statistical power and allow the full temporal trajectory of GlyNAC's effects to be characterized.

Acknowledgements. 

The authors thank Dr. D. Marmor, Mrs. N. Jaipershad, Dr. L. Wang, Ms. Zhu, Ms. Khemlani, Dr. J. Cohen, Dr. S. Lin, Mr. Z. Liang, and Ms. DePietro for their support and guidance throughout this research.

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