Cuprizone in Peanut Butter: An Alternative Method of Cuprizone Administration to Model Demyelination of the Central Nervous System

Abstract

Modeling disease in rodents is essential for preclinical research, particularly in the development of drugs and therapies. Traditional disease modeling approaches often rely on invasive and stressful procedures such as repeated injections and/or surgical approaches. These methods raise welfare concerns and can introduce stress-related variables that compromise model reliability. Cuprizone (CPZ) is a copper-chelating compound widely used to induce CNS demyelination in mice over a 6-week period via incorporation into ground rodent chow. However, the presumed adverse taste of CPZ reduces food intake, resulting in weight loss, inconsistent consumption, and variable levels of demyelination, complicating disease comparison. To address this, we proposed that mixing CPZ into peanut butter (CPZ-PB) would promote voluntary intake, standardize daily dosing, and minimize weight loss, while maintaining effective demyelination. Thirty mice were divided into control, CPZ-PB, or CPZ-chow groups and received treatment daily for 6 weeks. Body weight and CPZ intake were recorded daily. The effect of demyelination through mechanical allodynia was assessed via the dynamic plantar aesthesiometer. Immunohistochemical analysis of myelin proteins and astrocyte activation was conducted on the brain and spinal cord after 6 weeks of treatment. Voluntary consumption of CPZ-PB successfully ameliorated weight loss concerns and achieved consistent daily CPZ dosing. Behavioral and immunohistochemical analyses confirmed effective demyelination comparable to traditional CPZ-chow methods. This study introduces an improved CPZ administration strategy that enhances animal welfare and provides a more reliable model for demyelination research.

Introduction

The development of animal models for preclinical research has been fundamental to the advancement of drug and therapy discovery.¹ A wide range of animal models are used, generated using various techniques; however, most rely on invasive procedures such as surgeries and/or repeated injections. Consequently, these approaches can induce stress, pain, or discomfort, raising ethical concerns and potentially compromising the reliability of experimental outcomes.² This is particularly relevant for models requiring administration of insoluble compounds, or those with poor bioavailability, which often necessitate oral gavage, a technically demanding procedure associated with signs of significant animal stress such as increased anxiety-like behaviors and in some cases esophageal or gastric injury.^3–5 To address these limitations, alternative delivery methods have been explored to reduce animal stress while preserving effective disease modeling. Voluntary oral administration has emerged as a promising strategy, with studies demonstrating that these methods significantly lower stress responses as measured by corticosteroid levels compared with oral gavage or injectable routes, without compromising dosing efficacy.⁶ These methods incorporate compounds into palatable food vehicles such as peanut butter, strawberry jam, or cookie dough, facilitating consistent intake and improvements in animal welfare.^7–10

Cuprizone (CPZ), a copper-chelating compound, is widely used to model demyelination of the CNS in mice by selectively targeting mature oligodendrocytes, inducing their apoptosis after only a few days of treatment.^11–14 The cessation of CPZ treatment initiates a phase of regeneration in which spared endogenous oligodendrocyte precursor cells restore the oligodendrocyte population and initiate remyelination of neuronal axons.^15,16 As such, the CPZ model offers the ability to study both demyelination and remyelination within a single model system, providing a valuable platform for testing potential therapeutic treatments. The standard methodology of CPZ administration involves mixing 0.2%-0.3% CPZ into standard ground rodent chow for 6 or 12 weeks to achieve acute or chronic demyelination, respectively.^12,17 While effective, this method is associated with various issues that hinder study outcomes. The primary reported issue is substantial weight loss of around 10% from starting body weight,^18,19 a presumed direct result of reduced food intake, potentially due to the adverse taste of CPZ in the chow. In turn, as CPZ consumption is dependent on food intake, this leads to inconsistent daily dosing of CPZ. As each animal consumes a different quantity of food and therefore CPZ, there is a wide variability in CPZ consumption between animals, leading to variations in the level of demyelination.

To address these issues, groups have investigated alternative CPZ-administration methods such as the incorporation of CPZ into pelleted chow^20–23 or drinking water.²⁴ However, these studies reported reduced potency and efficacy in comparison to ground chow and failed to address the issues involving variations in CPZ dosing.^22,23 A potential reason for the failure of these methods is that CPZ is sensitive to the environment and is therefore degraded upon environmental exposure.¹³ In addition, as CPZ is a copper-chelator, when mixed into pelleted or ground chow, it is potentially inactivated due to prolonged interactions with the copper present in chow.¹¹ Successful CPZ intoxication via oral gavage has been demonstrated in both rats and mice; however, daily oral gavage for a 6- or 12-week period, as required for this model, would put extended stress on animals and therefore is not a feasible long-term alternative.^25,26

To improve CPZ administration and enhance animal welfare, we proposed mixing CPZ into peanut butter (CPZ-PB) as a palatable delivery vehicle, which would allow each animal to receive a standard daily dose to efficiently induce demyelination without the substantial weight loss seen with the traditional method of mixing CPZ into ground chow (CPZ-chow).

Ethical review.

All animal housing and procedures were designed in accordance with the New Zealand Animal Welfare Act 1999 and had approval from the University of Auckland Animal Ethics Committee.

Materials and Methods

Animals.

Thirty male C57BL/6J mice, aged 7 weeks and weighing 23.19 ± 1.39 g, were obtained from the Vernon Jansen Unit, University of Auckland. Animals were individually housed in blueline IVCs maintained in a 12-hour light-dark cycle with access to food and water ad libitum. All efforts were made to minimize the number of animals used and their suffering.

Study design.

The experimental timeline is illustrated in the schematic (Figure 1). Animals were randomly allocated into one of the following groups: control (n = 10), CPZ-PB (n = 10), or CPZ-chow (n = 10). Animals were acclimatized to their respective food consumption methods (ground chow from food hoppers or peanut butter) for 1 week before the onset of treatment. Body weight was measured and recorded for all animals daily throughout the entire study period. A priori power analysis was conducted using G*Power 3.1.9.7.²⁷ For immunohistochemical analysis, assuming an effect size of 1.6 based on previous studies conducted in our laboratory, an α error probability of 0.05, desired power of 0.8, and minimum total sample size of n = 9, with 3 treatment groups, we calculated the minimum sample size per group as n = 3. For dynamic plantar aesthesiometer analysis, assuming an effect size of 0.8, based on previous studies conducted in our laboratory, an α error probability of 0.05, desired power of 0.8, and minimum total sample size of n = 30, with 3 treatment groups, we calculated the minimum sample size per group as n = 10.

Figure 1.

Schematic Depiction of the Study Timeline. Thirty male, C57BL6J mice were divided into 3 groups: control, cuprizone and peanut butter (CPZ-PB), or CPZ-chow (n = 10 per group). A week before treatment, mice were acclimatized to either untreated peanut butter or untreated ground chow. Control animals were fed standard pelleted chow. CPZ-PB animals were fed with standard pelleted chow alongside 7.5 mg CPZ mixed into peanut butter for 6 wk. CPZ-chow animals were fed with 0.25% CPZ mixed into ground chow daily for 6 wk. Mechanical allodynia was assessed at day 0, 3 wk, and 6 wk with the dynamic plantar aesthesiometer (DPA). After 6 wk of treatment, animals were euthanized for immunohistochemical (IHC) analysis. Created with BioRender.com.

CPZ treatment.

The CPZ (bis[cyclohexane]oxalihydrazone; no. C9012; Sigma-Aldrich, St. Louis, MO) dosage was determined based on previous studies.²⁶ The CPZ chow mixture was prepared through mixing 0.25% CPZ into ground standard rodent chow (no. 2018X; Teklad, Inotiv, West Lafayette, IN), prepared fresh daily to avoid CPZ deactivation upon environmental exposure. Animals received 25 g of CPZ-chow mixture directly into food hoppers placed in individual cages; this was replaced daily for the 6-week study period. Smooth American Style Peanut Butter (PB) made up of roasted peanuts, sugar, hydrogenated vegetable oil (rapeseed oil, cottonseed oil, and soybean oil), salt, and molasses was used in this study. To generate CPZ-PB, 7.5 mg CPZ was mixed into 250 mg PB, a final concentration of 300 mg/kg for a 25-g mouse, dosing based on Zhen et al.²⁶ CPZ-PB was prepared fresh every 3 days and stored at 4 °C before treatment (Figure 2A). During treatment, animals were fed daily with a 250-mg pellet of CPZ-PB smeared onto the wall of the cage (Figure 2A). Daily CPZ consumption was recorded through measurements of remaining CPZ-chow or CPZ-PB.

Figure 2.

The Preparation and Consumption of CPZ-PB. (A) Preparation of CPZ-PB was achieved through mixing CPZ powder into peanut butter. CPZ-PB pellets were smeared onto the side of the cages for consumption. (B) Graph demonstrating CPZ consumption is more consistent when delivered in peanut butter. Data represent mean ± SEM (n = 10 per group). (C) Graph demonstrating animal body weight during study. Data represent mean ± SEM (n = 10 per group). Statistical significance was determined using a 2-way mixed ANOVA, and pairwise comparisons were performed using the Tukey post hoc test. ***P < 0.001 compared with control animals; ^#P < 0.05 compared with CPZ-PB animals.

Dynamic plantar aesthesiometer.

Mechanical allodynia was quantitatively assessed using the automated dynamic plantar aesthesiometer (DPA; 37550; Ugo Basile, Gemonio, Italy) at day 0 (baseline), 3 weeks, and 6 weeks of CPZ treatment. Briefly, mice were placed into elevated transparent plastic chambers on a wire mesh platform for 30 minutes to allow for acclimatization. The DPA filament was placed underneath the mesh platform at a 90° angle to the plantar surface of the hind paw. The pressure of the filament increased toward the paw until a withdrawal response was elicited at which point the force and threshold values were recorded.²⁸ Each paw was measured in triplicate and averaged per mouse by a blinded investigator.

Tissue collection and processing.

After 6 weeks of CPZ treatment, mice were euthanized with an intraperitoneal injection of pentobarbtial (0.25 mg/100 g) and transcardially perfused with 0.9% saline followed by 4% paraformaldehyde (PFA). Whole brains and spinal cords were extracted and stored in PFA overnight for postfixing. Tissues were then cryopreserved with incubations in 30% sucrose, 50% sucrose, and finally an ethylene glycol-based cryoprotectant. The brain was sectioned coronally in 30-µm sections using a sliding microtome (SM2010R; Leica, Wetzlar, Germany), and the spinal cord was sectioned longitudinally in 15-µm sections using a freezing cryostat (CM3050S; Leica, Wetzlar, Germany) and mounted onto Superfrost slides (no. 71869-10; Electron Microscopy Sciences, Hatfield, PA). Demyelination was assessed in the midbody of the corpus callosum, excluding the genu and splenium of the bundle. The extent of demyelination was assessed by quantification of fluorescence intensity of myelin expression using myelin basic protein (MBP) (MAB386, antirat; 1:100; Millipore Sigma, Auckland, New Zealand) and myelin oligodendrocyte glycoprotein (MOG) (ab233549, antirabbit; 1:250; Abcam, Cambridge, England). The astrocyte response was assessed by quantification of fluorescence intensity of glial fibrillary acidic protein (GFAP; no. Z0334, antirabbit; 1:1,000; Sigma-Aldrich, St. Louis, MO). Brain sections were stained free-floating, while spinal cord sections were stained directly on Superfrost slides. Sections were incubated in primary antibody solution containing the primary antibody diluted in PBS with 3% normal goat serum (NGS; 7481; Abcam, Cambridge, England), overnight at 4 °C. Sections then underwent a 4-hour incubation in secondary antibody solution containing secondary antibody diluted in PBS with 3% NGS at room temperature. The species-appropriate Alexa Fluor conjugated secondary antibodies goat anti-rat IgG 488 (no. A-11006; 1:500; Invitrogen, Thermo Fisher Scientific, Waltham, MA) and goat anti-rabbit IgG 594 (no. A-11012; 1:500; Invitrogen, Thermo Fisher Scientific, Waltham, MA) were used to fluorescently label the antigens of interest MBP, MOG, and GFAP, respectively. Sections were imaged using a Nikon TE2000E inverted microscope (TE2000E; Nikon Instruments, Melville, NY). The extent of MBP, MOG, and GFAP staining was assessed by measuring the integrated density in ImageJ, following consistent background subtraction and threshold adjustment from 3 images captured throughout the corpus callosum of brain sections and 5 images captured from the white matter down the length of spinal cord sections to ensure each spinal cord level/region was accounted for. The total MBP+, MOG+, and GFAP+ density was calculated for each animal (n = 4 sections per animal), and an average per treatment group was calculated (n = 3 animals per group).

Statistical analysis.

Mouse body weight over the course of the study was recorded and reported as a change in body weight from day 0. Statistical analysis was performed using SPSS Statistics (IBM, Armonk, NY). Statistical significance was determined using a 2-way mixed ANOVA and Tukey post hoc test. DPA data were reported as the mean force at paw withdrawal, and statistical significance was determined using raw values and a 2-way mixed ANOVA followed by Bonferroni post hoc analysis. The MBP+, MOG+, and GFAP+ fluorescence data were reported as a percentage of the fluorescence intensity of untreated animals. Statistical significance was determined using raw values and a one-way ANOVA followed by Bonferroni post hoc analysis. Normality of the data was first confirmed using the Shapiro-Wilk test. All other assumptions for conducting either the one-way ANOVA or 2-way mixed ANOVA were also verified before running the tests.

Results

Administration of CPZ in peanut butter achieves standardized daily dosing and ameliorates initial weight loss.

Mixing CPZ into peanut butter encouraged voluntary uptake of CPZ, masking the possible adverse taste of CPZ. On average, CPZ-chow animals consumed 8.33 ± 0.38 mg of CPZ daily, while the CPZ-PB animals consumed 7.26 ± 0.04 mg daily (Figure 2B). Daily recordings of CPZ consumption demonstrate more consistent dosing with CPZ-PB in comparison to CPZ-chow, which fluctuates throughout the study period (Figure 2B). The body weight of each animal was measured daily over the study period to determine the effect of CPZ treatment. Control animals displayed a steady increase in body weight of 12.7% ± 0.92% from starting weight to the end of the study (Figure 2C). CPZ-PB animals showed a similar trend with a 6.5% ± 0.78% increase. Conversely, CPZ-chow animals demonstrated a significant reduction in body weight of 10% ± 1.05% (P < 0.001) over the study period (Figure 2C). This reduction in body weight was evident immediately after the onset of CPZ treatment with a 5.7% ± 3.15% reduction in body weight of CPZ-chow animals after only 1 weeks of treatment, an effect not seen in CPZ-PB animals. Over the 6-week study period, in comparison to control animals, CPZ-chow animals displayed a significant reduction in weight from day 17 to day 47 (day 17, P < 0.001; day 47, P < 0.001). CPZ-PB animals also showed a reduction in weight gain compared with controls; however, this effect was only significant at the later time point of day 36 (P = 0.030). Comparing the CPZ-treatment groups to each other, CPZ-chow animals displayed a significant reduction in weight from day 17 in comparison to CPZ-PB animals (P = 0.016) (Figure 2C). Taken together, this indicates that daily treatment of CPZ in chow is associated with severe weight loss, an effect previously documented within the literature.^21,29 However, this can be ameliorated by administration of CPZ in peanut butter, which provides more consistent dosing without severe weight loss.

Administration of CPZ in peanut butter increases mechanical allodynia to a greater extent than CPZ-chow-treated animals.

Demyelination of the CNS is associated with increased mechanical allodynia, which can be quantitatively assessed using the automated DPA. We assessed mechanical allodynia using the DPA at day 0 (baseline), 3 weeks, and 6 weeks of CPZ treatment. As expected, untreated animals displayed no change in the mean withdrawal response threshold over the study period. CPZ treatment induced changes to this response with CPZ-chow animals displaying a significant reduction in the withdrawal response threshold from baseline to 3 weeks of treatment (P = 0.012) as well as at 6 weeks of treatment (P = 0.044). CPZ-PB animals displayed a significant reduction in the withdrawal response from baseline at 6 weeks of treatment (P = 0.003). These data suggest CPZ-chow treatment induces a steady onset of mechanical allodynia. However, in comparison to untreated animals, after 3 weeks of treatment, only CPZ-PB animals displayed a reduction in withdrawal response threshold indicating the onset of mechanical allodynia (P = 0.025). At 6 weeks of treatment, both CPZ-PB and CPZ-chow-treated animals displayed a reduction in withdrawal response in comparison to untreated animals, an effect that is greater in CPZ-PB animals than in CPZ-chow animals (CPZ-PB; P < 0.001; CPZ-chow; P = 0.030) (Figure 3). While the induction of mechanical allodynia in mice through CPZ treatment is an expected response, these data demonstrate that CPZ-PB treatment produces an earlier and more significant behavioral deficit.

Figure 3.

Administration of CPZ in Peanut Butter Reduces Mechanical Sensitivity Threshold to a Greater Extent than CPZ-Chow-Treated Animals 6 wk Posttreatment. Mechanical allodynia was assessed at baseline (0 wk), 3 wk, and 6 wk posttreatment via the DPA. Data represent mean ± SEM with n = 10 per group. Statistical significance was determined using a 2-way mixed ANOVA, and pairwise comparisons were performed using the Bonferroni post hoc test. *P < 0.05, ***P < 0.001 for CPZ treatment compared with control; ^#P < 0.05, ^##P < 0.01 for effect of time within treatment group.

Administration of CPZ in peanut butter induces demyelination and astrocyte activation in both the brain and spinal cord.

Brain and spinal cord tissue was evaluated via immunohistochemical analysis of the myelin proteins MBP and MOG, and GFAP to examine the effect of CPZ treatment on myelination and astrocyte processes, respectively. Quantification of MBP fluorescence intensity within the corpus callosum of the brain revealed a significant decrease in expression following CPZ treatment in both treatment groups (CPZ-PB, P < 0.001; CPZ-chow, P < 0.001) (Figure 4A-C). This was an effect also seen with MOG fluorescence intensity measurements. However, CPZ-PB animals displayed a greater reduction in MOG expression (P = 0.008) compared to controls than did CPZ-chow animals (P = 0.014) (Figure 4D-F). GFAP staining in the brain revealed a localized presence of GFAP+ astrocytes in the corpus callosum of the brain (Figure 4G). CPZ-treated animals appeared to have an increase in GFAP+ expression in both the corpus callosum and surrounding cortex (Figure 4G). Quantification of GFAP fluorescence intensity within the corpus callosum confirmed this, revealing a significant increase in the expression of GFAP following CPZtreatment in both groups (CPZ-PB; P < 0.001; CPZ-Chow; P < 0.001) (Figure 4H).

Figure 4.

CPZ-PB Treatment Induces Demyelination and Astrogliosis in the Corpus Callosum of the Brain. (A and B) Myelin basic protein (MBP) and (D and E) myelin oligodendrocyte glycoprotein (MOG) expression in the brain show the myelinated tracts of the corpus callosum. (A and D) Scale bar: 500 mm; (B and E) scale bar: 250 mm. Quantification of the mean (C) MBP and (F) MOG fluorescence intensity within the corpus callosum in untreated, CPZ-PB, and CPZ-chow animals. Data represent mean ± SEM with n = 3 per group. Statistical significance was determined using a one-way ANOVA, and pairwise comparisons were performed using a Bonferroni post hoc test. *P < 0.05, **P < 0.01 and ***P < 0.001. (G) Glial fibrillary acidic protein (GFAP) expression in the brain. Scale bar: 500 µm. (H) Quantification of the mean GFAP fluorescence intensity within the white matter of the spinal cord in untreated, CPZ-PB, and CPZ-chow animals. Data represent mean ± SEM with n = 3 per group. Statistical significance was determined using a one-way ANOVA, and pairwise comparisons were performed using the Bonferroni post hoc test. ***P < 0.001.

Spinal cord tissue showed abundant expression of myelin proteins MBP and MOG throughout the white matter (Figure 5A and C). Quantification demonstrated a significant reduction in the expression of both MBP (CPZ-PB, P < 0.001; CPZ-chow, P < 0.001) and MOG (CPZ-PB, P < 0.001; CPZ-chow, P < 0.001) in CPZ-PB and CPZ-chow animals compared with controls (Figure 5B and D). GFAP staining of spinal cord tissue appeared to be localized to white matter regions and increased in animals treated with CPZ (Figure 5E). Quantification demonstrated a significant increase in GFAP expression in the white matter following CPZ treatment (CPZ-PB, P < 0.001; CPZ-chow, P < 0.001) (Figure 5F). Combined, these results demonstrate that administration of CPZ in peanut butter is effective at inducing demyelination in both the brain and spinal cord to levels comparable to the gold-standard CPZ-chow method.

Figure 5.

CPZ-PB Treatment Induces Demyelination of White Matter (WM) in the Spinal Cord as Well as an Astroglial Response. (A) MBP and (C) MOG expression in the spinal cord show the longitudinal myelinated tracts of the white matter. Scale bar: 150 mm. GM, gray matter. Quantification of the mean (B) MBP and (D) MOG fluorescence intensity within the white matter of the spinal cord in untreated, CPZ-PB, and CPZ-chow animals. Data represent mean ± SEM with n = 3 per group. Statistical significance was determined using a one-way ANOVA, and pairwise comparisons were performed using the Bonferroni post hoc test. ***P < 0.001. (E) Glial fibrillary acidic protein (GFAP) expression in the spinal cord. Scale bar: 250 µm. (F) Quantification of the mean GFAP fluorescence intensity within the white matter of the spinal cord in untreated, CPZ-PB, and CPZ-chow animals. Data represent mean ± SEM with n = 3 per group. Statistical significance was determined using a one-way ANOVA, and pairwise comparisons were performed using the Bonferroni post hoc test. ***P < 0.001.

Discussion

Traditional administration of the compound CPZ to model demyelination of the mouse CNS has been questioned due to issues such as substantial weight loss, variability in CPZ consumption, and potential CPZ degradation upon environmental exposure, all of which complicate its use. In this study, we present an improved method of CPZ administration through incorporating CPZ into peanut butter to facilitate voluntary oral uptake. This approach effectively mitigated the weight loss typically observed when CPZ is delivered via ground chow.^21,29 CPZ-PB-treated animals maintained expected body weight throughout the experimental period, likely due to access to unaltered standard chow and the calorific support of peanut butter. In contrast, animals treated with CPZ mixed into ground chow exhibited a progressive reduction in body weight. This highlights a major benefit of our methodology, as ethical endpoints in animal studies are often based on weight loss thresholds, beyond which animals are either taken off treatment or excluded from study, both of which hinder study outcomes.

Incorporation of CPZ into peanut butter enabled standardization of daily dosing, delivering 7.5 mg of CPZ to each mouse daily over a 6-week period. This was a dosage based on CPZ administration via oral gavage, described by Zhen et al,²⁶ using 300 mg/kg for an average 25-g mouse. Although our study used a fixed dose of 7.5 mg, this methodology allows for easy adjustment of dosage based on individual body weight, further reducing dosing variability between animals. This is a considerable advantage over traditional CPZ-chow methods, which often require specialized feeding apparatuses that are prone to tipping over and spilling feed. This interferes with the accurate measurement of food and CPZ consumption.³⁰ In addition, CPZ-chow methods typically require housing mice individually, potentially impacting normal social behaviors, and the absence of pelleted chow prevents the ability for mice to naturally file down their incisors.^30,31

After confirming efficient and standardized uptake of CPZ-PB without substantial weight loss, we next assessed mechanical allodynia, a response linked to demyelination of the CNS.^32,33 Demyelination disrupts the myelin sheath of nerves, impairing nerve cell communication, and is associated with mechanical allodynia, a heightened pain perception in response to a normally innocuous stimulus.³⁴ Studies using CPZ to induce demyelination in animal models have indicated the onset of neuropathic pain and allodynia.^35,36 Through DPA testing, we demonstrated that CPZ-PB treatment induces mechanical allodynia earlier, with initiation at 3 weeks of administration, and to a greater extent than CPZ-chow treatment by 6 weeks of treatment. These findings suggest that the consistent and effective daily dosing provided by CPZ-PB may enhance demyelination induction in mice.

To confirm demyelination, brain and spinal cord tissue was assessed for the expression of myelin markers MBP and MOG at 6 weeks of treatment. The corpus callosum is a heavily myelination region of the brain, known to be affected by CPZ treatment,²¹ and was therefore the region analyzed. Immunohistochemical analysis revealed a significant reduction in MBP and MOG expression in the midbody of the corpus callosum following CPZ treatment in comparison to untreated controls. The spinal cord is also known to be affected by CPZ treatment; however, this effect is not often reported, with some studies reporting a lack of demyelination within the cord.^11,37 Immunohistochemical analysis of spinal cords in the current study demonstrated that demyelination does occur, as measured by a significant reduction in MBP and MOG expression within the white matter of the spinal cord. These analyses further confirmed CPZ-PB as an effective method for delivering CPZ while still maintaining demyelination in both the brain and spinal cord. To extend these findings, we assessed the activation of astrocytes in response to CPZ treatment. The activation of astrocytes, as commonly measured by increased expression of GFAP, has been widely linked to the general onset and maintenance of neuropathic pain.³⁸ In line with this, our analysis revealed a significant increase in GFAP fluorescence intensity within both the corpus callosum of the brain and the white matter of the spinal cord in animals treated with CPZ. No difference in the mechanism of CPZ intoxication was noted. Taken together, these findings indicate that CPZ delivery in PB induces a comparable astrocyte response to when CPZ was administered in chow. This glial response may contribute to the mechanisms underlying mechanical allodynia; however, future work should be conducted to investigate microglial and other biologic processes that may relate to mechanical allodynia to further link CPZ treatment to causation.

This study utilized peanut butter as a delivery vehicle to standardize CPZ dosing in mice over a 6-week period. While we report standard and consistent delivery, in some parameters, we report enhanced demyelination and behavioral responses indicative of demyelination in animals treated with CPZ-PB. While this could be a direct result of more efficient CPZ intoxication, it cannot be overlooked that a potential mechanism occurring here is an indirect effect of peanut butter enhancing the bioavailability of CPZ. It has been well documented that the use of food platforms such as oil-water emulsions or bioenhancers such as piperine and quercertin enhance the bioavailability of drugs.^39,40 Hence, whether peanut butter as a delivery vehicle enhances the bioavailability of CPZ requires further evaluation. While we demonstrate that peanut butter is effective at inducing voluntary uptake of CPZ, it is important to consider the impact of daily peanut butter consumption on mice over a 6-week period. We saw no increase in the body weight of animals receiving CPZ-PB; however, it is likely that for longer term studies there may be negative impacts of consistent peanut butter consumption due to fat content, which would require further investigation outside the scope of this study. Nevertheless, this study identifies and supports the concept of using food delivery vehicles for administering CPZ, indicating the potential for different vehicles such as jelly or jam of which the nutritional content could perhaps be more strictly controlled. In addition, in this study, we used male C57/BL6J mice, the predominant species and strain used for CPZ modeling¹³; however, we suggest that the use of food delivery vehicles for drug and/or compound administration can be extended to other species and strains.

Importantly, this study addresses the 3Rs of animal use in research, replacement, reduction, and refinement, a key set of principles to minimize harm and improve animal welfare.⁴¹ The ability to standardize daily dosing decreases interindividual variability, which in turn will decrease the sample size needed to achieve the same statistical power, ultimately reducing the number of animals required for a study. Furthermore, the use of peanut butter as a delivery vehicle reduces the direct effect on the animal, removing the need to modify or reduce a typical food source, an effect we directly demonstrate that correlates with no negative changes to weight.

We present a novel method of CPZ administration that standardizes daily dosing over a 6-week period without causing significant weight loss. This approach results in increased mechanical allodynia, a process indicative of CNS demyelination, which has been confirmed through immunohistochemical analysis of myelin expression in the brain and spinal cord, as well as expected astrocytic responses. Future studies should extend this work, investigating the expression of immature oligodendroglial markers, mature myelin markers, and microglial markers, as well as look to extend the timeframe to model chronic demyelination over a 12-week period, with an included analysis that assesses the remyelination profile upon cessation of CPZ treatment. In addition, studies should be conducted in female mice to investigate if our findings are gender specific or if PB can be used as a delivery vehicle for all animals irrespective of gender. Taken together, we propose the use of peanut butter as a delivery vehicle for CPZ administration as it promotes voluntary oral uptake, minimizes weight loss, and most importantly addresses key animal welfare concerns.

Conflict of Interest

The authors have no conflict of interest to declare.

Funding

This research was supported by the New Zealand Multiple Sclerosis Research Trust. B.H. is funded by the Neurological Foundation of New Zealand Gillespie Doctoral Scholarship.

Author Contributions

Brooke Hawker; Data curation, Formal analysis, Investigation, Methodology, Writing – original draft. Ethan Chiddicks; Data curation, Investigation. Jack Flanagan; Conceptualization, Supervision Bronwen Connor; Conceptualization, Supervision, Writing – review & editing. Amy McCaughey-Chapman; Conceptualization, Investigation, Methodology, Supervision, Writing – review & editing.

Data availability

Data are available upon reasonable request.