Introduction
Diabetes mellitus (DM) is a chronic metabolic disease characterized by elevated blood glucose levels due to insufficient insulin secretion or impaired insulin action. According to the International Diabetes Federation, approximately 537 million adults worldwide have diabetes, and this number is projected to increase to 783 million by 2045 [1]. Beyond glycemic control, DM is closely associated with various complications. Among these, diabetic peripheral neuropathy (DPN) is a common neurological complication that causes sensory abnormalities through peripheral nerve damage, affecting 30–50% of diabetic patients. The main symptoms include pain, tingling, and sensory loss [2,3]. Particularly, neuropathic pain and affective disturbances such as depression are representative comorbidities frequently reported in DPN [4].
Animal models are essential tools for investigating the mechanisms underlying these comorbidities and developing therapeutic strategies. The streptozotocin (STZ)-induced diabetic model has been widely used, with drugs such as dihydromyricetin, palmatine, and hesperidin reported to exhibit both analgesic and antidepressant effects in this model [5,6]. These studies have mainly employed behavioral paradigms such as the von Frey test and the forced swim test (FST) to evaluate pain and depression. However, previous studies using STZ models have shown inconsistent findings regarding the development of depressive behaviors after STZ injection. For instance, some studies reported increased immobility time in the tail suspension test (TST) as early as the first week, while others observed reduced immobility, delayed onset, or no significant changes at all [7-9]. These inconsistencies may be attributed not only to differences in STZ dose and administration protocols [10] but also to variations in the timing and methods of affective behavior assessment. This suggests the need for more diverse behavioral assessment models when evaluating affective disturbances in diabetic animals [11]. The marble burying test (MBT) is widely used as a behavioral paradigm reflecting repetitive digging behavior and can assess anxiety- or depression-like tendencies [12-15].
In this study, we aimed to comprehensively characterize behavioral changes associated with mechanical allodynia and affective alterations in STZ-induced diabetic mice. Specifically, we evaluated mechanical sensitivity using the von Frey test and assessed anxiety-like behaviors using the elevated plus maze (EPM) and open field test (OFT), and depression-like behaviors using the TST and MBT. Through this multi-faceted behavioral assessment approach, we aimed to establish a more reliable and valid evaluation model for studying affective disturbances in diabetic conditions.
Materials and Methods
1. Animals
The experiment was conducted using 12-week-old male C57BL/6 mice (25–30 g; DBL). Mice were housed in cages with free access to food and water, maintained under a controlled environment at room temperature (23 ± 2℃) with a 12/12-hour light/dark cycle. All experimental procedures were performed in accordance with the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Kyung Hee University (IACUC approval number: KHSASP-24-168), following the National Institutes of Health (NIH) guide for the Care and Use of Laboratory Animals (NIH publication no. 86- 23, 1985).
2. Diabetes induction
Diabetes was induced in mice using STZ (Sigma Chemical Co.) dissolved in 0.1 M sodium citrate buffer (pH 4.5) at a dose of 150 mg/kg body weight via single intraperitoneal injection. Adult male C57BL/6 mice were randomly assigned to either the control (vehicle) group or the STZ-treated group prior to the experiment. Randomization was performed using a simple random allocation method to ensure unbiased group assignment. After STZ administration or vehicle injection, diabetes induction was confirmed by blood glucose measurements, and only mice with blood glucose levels exceeding 300 mg/dL were included in the STZ group for subsequent behavioral analyses. Blood glucose and body weight were measured weekly for 1 and 2 weeks post-STZ injection using a glucometer (Accu-Chek Active; Roche Diabetes Care).
3. Mechanical allodynia assessment
Mechanical allodynia was assessed using the up-down method to determine the 50% withdrawal threshold. Mice were placed individually in plastic cages on a wire mesh floor and allowed to acclimate before testing. Von Frey filaments (North Coast Medical) of varying stiffness were applied perpendicular to the plantar surface of the hindpaw to induce mechanical stimulation. The logarithmic incremental bending forces (0.008, 0.02, 0.04, 0.07, 0.16, 0.4, 1.0, and 1.4 g) were applied sequentially, and a positive response was defined as rapid paw withdrawal or licking behavior. Starting with a baseline force of 0.008 g, the filament was applied to the center of the hindpaw for 6 seconds, and subsequent filaments were selected using the up-down method based on the response. The 50% mechanical withdrawal threshold was calculated using this method. Measurements were taken at baseline (before STZ injection), and at 1 and 2 weeks post-injection, with mechanical sensitivity assessed using the same procedure.
4. Behavioral testing
1) Elevated plus maze
Mice were allowed to freely explore for 5 minutes on an apparatus elevated 50 cm above the floor, consisting of two open arms (30 × 6 cm) and two closed arms (30 × 6 × 15 cm) arranged in a cross shape perpendicular to each other, with a central zone (6 × 6 × 15 cm). Time spent in the open and closed arms was recorded using the SMART system (V3.0; Panlab). Since the black floor of the apparatus prevented proper mouse detection, white tape was applied to the floor to prevent recognition errors.
2) Open field test
Locomotor activity and exploratory behavior were recorded for 10 minutes in a white square arena (40 × 40 × 40 cm) using the SMART system (V3.0). Total distance traveled and time spent in the center zone were calculated and used as indicators of locomotor activity and anxiety- or depression-like behaviors.
3) Tail suspension test
Adhesive tape was cut to a total length of 17 cm and attached to the tail 2 cm from the tip. To prevent mice from climbing up their tails, a custom-made climb stopper (height 2.5 cm, open on both sides) was fitted onto the tail. The tail was secured to a flat plate connected to a horizontal metal bar, and mice were suspended for 6 minutes. Immobility time was manually recorded using video recording (HDR-CX280; SONY). Immobility duration was measured using a stopwatch (XL-013 Any Time; I&People Co.).
4) Marble burying test
Mice were observed for 5 minutes of free behavior in a cage containing glass marbles arranged in 4 rows and 5 columns. After the test, the number of marbles buried more than twothirds in the bedding was counted [16-18].
5. Statistical analysis
Data are expressed as mean ± standard error of the mean. Two-way ANOVA followed by Bonferroni post-hoc test was used for repeated measures (blood glucose, body weight, and mechanical allodynia), and unpaired Student’s t-test for single time-point behavioral assessments (EPM, OFT, TST, and MBT). Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software), and p ＜ 0.05 was considered statistically significant.
Results
1. Hindpaw mechanical allodynia in the diabetic mouse model
Fasting blood glucose levels in the STZ-treated group were significantly elevated compared to the control group at 1 week post-injection and remained consistently hyperglycemic at the 2-week time point (Fig. 1A). Body weight showed a decreasing trend following STZ administration (Fig. 1B). The mechanical withdrawal threshold in the hindpaw was significantly reduced in the STZ-treated group from 1 week post-injection and remained at a low level through the 2-week time point (Fig. 1C). These findings confirm that the STZ diabetic model successfully induced typical diabetic symptoms and mechanical allodynia in the peripheral region.
2. Anxiety- and depression-like behaviors in the elevated plus maze
In the EPM, the STZ group showed a tendency toward reduced time spent in the open arms compared to the control group, but this did not reach statistical significance (Fig. 2A). Time spent in the closed arms also showed an increasing trend compared to the control group, but no significant difference was observed (Fig. 2B).
3. Behavioral changes in the open field test
In the OFT, the STZ group showed significantly reduced time spent in the center zone compared to the control group (Fig. 3A), while time spent in the peripheral zone was significantly increased (Fig. 3B). Total distance traveled was also significantly reduced compared to the control group (Fig. 3C).
4. Depression-like behavior in the tail suspension test
In the TST, the immobility time in the STZ group was not significantly different from the control group, but showed an increasing trend (Fig. 4).
5. Anxiety- and depression-related behaviors in the marble burying test
In the MBT, the control group buried a large number of marbles, resulting in a high average count, whereas the STZ group showed a markedly reduced number of buried marbles (Fig. 5). This suggests that anxiety- and depression-like behaviors can develop in the STZ-induced diabetic state.
Discussion
In this study, we aimed to establish a mouse model exhibiting both diabetic neuropathic pain and depression-like behaviors by evaluating metabolic parameters, hindpaw mechanical allodynia, and various affective behavioral tests in STZ-induced diabetic mice. The STZ-treated group demonstrated sustained hyperglycemia, weight loss, and mechanical allodynia in the hindpaw. Although affective behavioral changes in the EPM and TST were modest, reduced central exploration time in the OFT and decreased marble burying behavior in the MBT were observed. These findings are consistent with previous studies demonstrating that STZ-induced diabetic rodent models exhibit not only metabolic abnormalities but also alterations in pain and affective behaviors [19]. The sustained elevation in blood glucose and weight loss observed following STZ administration represent typical metabolic characteristics of the type 1 diabetes model and have been previously reported [20,21]. Our study similarly demonstrated these metabolic abnormalities along with a significant reduction in hindpaw mechanical withdrawal threshold, indicating sustained mechanical allodynia. Furthermore, we confirmed the comorbidity of depressionlike behaviors associated with pain. This suggests that the STZ-induced diabetic mouse model can be utilized not only for pain research but also for investigating the pathophysiology of pain-depression comorbidity.
Regarding affective behaviors, the STZ group showed trends toward reduced time spent and entries in the open arms of the EPM, although these changes were less pronounced compared to those observed in the OFT. Previous studies have also reported that anxiety-related measures in the EPM are inconsistent in STZ models depending on experimental conditions, with some studies indicating that while depression indices in the TST and the FST are robust, EPM findings are mild or nonsignificant [20]. This suggests that diabetes may influence depression- related behaviors more strongly than typical anxiety indices based on open spaces and elevated platforms, or that sensitivity may vary depending on test conditions. Himanshu et al. [22] suggested that assessing anxiety in diabetic models requires multiple tests, such as the hole-board test and lightdark box test, rather than relying on the EPM alone. Meanwhile, the STZ group showed reduced center zone duration and total distance traveled in the OFT, and although not statistically significant, showed a trend toward increased immobility time in the TST. Castillo-Gómez et al. [7] reported that increased TST immobility time in STZ diabetic mice was accompanied by structural and synaptic plasticity changes in prefrontal cortex interneurons, while Miyata et al. [8] and Bampi et al. [9] also interpreted increased immobility in the TST and FST as depression-like behavior in STZ-induced diabetic mice. Other studies in STZ models have reported reduced total distance traveled and decreased center zone time in the OFT, which were associated with overall decreased activity and increased depression levels [21]. In our study, the OFT results showed significant changes, while the TST showed trends. These findings support the notion that the STZ-induced diabetic model can function as a model exhibiting depression-like behaviors in addition to pain.
The significantly reduced number of buried marbles in the STZ group compared to controls in the MBT was particularly interesting. While the MBT is typically used as an indicator of anxiety or compulsive-like behavior, recent studies have proposed new interpretations suggesting it may reflect defensive (neophobic/defensive) behavior and repetitive, perseverative behaviors. Thomas et al. [16] and de Brouwer et al. [15] specifically noted that the MBT should not simply be interpreted as “more burying equals higher anxiety,” but rather that both excessive and reduced burying behaviors can indicate abnormal behavior depending on the context. Therefore, in our study, given the reduced total distance traveled in the OFT in the STZ group, the decreased marble burying may reflect reduced activity rather than decreased compulsive behavior.
Our study has several limitations. First, some clinical studies have reported sex-dependent differences in sensitivity to diabetic neuropathy and depression [23]. Therefore, additional experiments using female mice are needed to evaluate sex differences. Second, although we observed behavioral changes associated with diabetes-related pain, we did not perform cellular and molecular analyses such as GFAP and Iba-1 expression in emotion-related brain regions including the hippocampus, prefrontal cortex, and amygdala. Several previous studies, including those by Castillo-Gómez et al. [7] and Miyata et al. [8], have reported that inflammatory markers are closely associated with depression-like behaviors in STZ diabetic models. These aspects will be addressed in future studies.
In conclusion, previous studies indicate that different behavioral tests reflect distinct affective constructs such as anhedonia, behavioral despair, and anxiety, and often yield inconsistent results depending on the test even within the same pain model [24,25]. Therefore, assessing pain-depression comorbidity requires multiple affective behavioral tests rather than relying on a single behavioral paradigm. Our findings demonstrate that the STZ-induced diabetic mouse model exhibits both peripheral mechanical allodynia and depression-like behavioral alterations, with outcomes varying across different assessment paradigms. This model may serve as a valuable tool for investigating the mechanisms underlying pain-depression comorbidity associated with diabetes.
