Behavioral Assessment of Stress Compensation in Minipigs Transgenic for the Huntington Gene Using Cortisol Levels: A Proof-of-Concept Study

Abstract

Background:

Huntington’s disease (HD) is an autosomal-dominant, progressive neurodegenerative disorder with motor, cognitive, behavioral and metabolic symptoms. HD patients exhibit an altered response to stress which is reflected in changes of cortisol levels. Large animal models of HD such as the Libechov minipig are currently explored in preclinical studies to improve translational reliability and assessing behavior is of interest.

Objective:

This study aimed to investigate whether cortisol metabolism and response to stress are changed in minipigs transgenic for the Huntington gene (tgHD) compared to wildtype (wt) animals suggesting that cortisol may be used as a marker for stress in minipigs.

Methods:

Thirty-two Libechov minipigs (14 tgHD and 18 wt) were tested before, during and after a stressor, i.e., a hoof trimming procedure, was applied at baseline and after one year. A total of six saliva samples were collected at each assessment and cortisol was measured. In addition, body temperature and respiratory rate were assessed at three pre-determined points during each hoof trimming procedure.

Results:

All minipigs showed a rise of cortisol in response to the hoof trimming stressor similarly to cortisol changes induced by stress observed in humans. No relevant differences were detected between tgHD and wt minipigs.

Conclusion:

Cortisol testing for the assessment of stress compensation, e.g., during hoof trimming, is feasible and well tolerated in wt and tgHD minipigs. To elucidate the time profile of cortisol responses to stressors further studies with assessments at multiple time points and exploration of the diurnal profiles of cortisol in minipigs are recommended.

Keywords

Animal models minipigs phenotyping biomarker saliva cortisol vital signs

INTRODUCTION

Huntington’s disease (HD) is a neurodegenerative genetic disorder caused by an autosomal dominant mutation in the Huntingtin (HTT) gene [1]. An extended poly-glutamine (poly-Q) section in the HTT protein is translated to an abnormal CAG repeat length (≥36) in the HTT gene and likely induces a toxic gain-of-function [2]. HD is characterized by motor and cognitive dysfunction as well as behavioral and psychiatric changes, e.g., depression [1]. Typically, behavioral and psychiatric pathology, particularly depression, alter responses to stress and thus stress plays a major role in various pathophysiological processes associated with neurodegenerative diseases and mental disorders [3 –5].

Stress increases the activity of the hypothalamic-pituitary-adrenal (HPA) axis, a pathway commonly involved in stress-response across species. Consecutive increases in the synthesis of the stress hormone cortisol (glucocorticoid) [6], regulated by the HPA-axis [7], are observed. Elevated HPA-axis activity is seen in patients suffering from depression [8]. In R6/2 HD mice treatment with high doses of corticosterone (rodent homolog of cortisol) resulted in weight loss and shortened lifespan [9]. Studies with rodents and primates exposed to stress revealed neuronal loss in the hippocampus and atrophy and volume reduction in the prefrontal cortex [10 –12]. Neurodegeneration in humans has also been found in the hypothalamus [13]. These findings suggest that prolonged exposure to stress, i.e., cortisol, enhances neurodegenerative processes in the brain [14, 15]. Therefore, monitoring of cortisol levels and responses seem reasonable and cortisol now is accepted and routinely used as a marker to assess response to stress [16].

In fact, cortisol metabolism was shown to be altered in human HD. In some studies of symptomatic HD patients plasma cortisol levels were increased and increased salivary cortisol was detected in pre-manifest HD gene carriers [17, 18]. While this was in line with clinical expectations a study in another patient cohort reported hypocortisolism in early-HD [19].

Psychiatric symptoms, such as depression, are a key driver of functional impairment in HD patients [20]. Therefore, methods assessing this domain in HD animal models are desirable for preclinical studies. Assessing psychiatric pathology in animal models behaviorally can be a challenge. We recently established a battery of motor, cognitive and behavioral tests to phenotype transgenic (tg) HD minipigs (124Q N-terminal fragment model) [21]. Establishing an objective marker for behavioral changes such as cortisol could supplement available methods and serve as a welcome and easy to apply option for future preclinical studies with minipigs.

The normal human diurnal cortisol profile shows increased levels shortly after awakening and a subsequent decrease during the rest of the day [22]. Of note, cortisol peaks are commonly observed approximately 30 minutes after stressors are applied [16 , 23–25]. The tgHD R6/1 pre-motor onset female mice also demonstrated an increased corticosterone release after exposure to a stressor [26]. Similar peaks were reported in pig salivary cortisol levels when food/water restriction or transport simulation were applied as stressors [27]. We concluded that this method might be applicable to assess cortisol levels in tgHD minipigs non-invasively and serve as surrogate for stress compensation and management.

In order to provide a standardized environment and stressor, we took advantage of the minipigs regular need for hoof trimming. Since the minipigs were housed on straw to provide animal-friendly conditions, excessive growth of hoofs was observed regularly. A custom-made hoof trimming cage was designed. Minipigs were suspended in this cage to perform hoof trimming, which resulted in unavoidable stress for the animals by [1] exposure to the setup, [2] entering the cage, [3] being lifted without contact to the ground, and [4] the hoof trimming procedure.

Hence, we decided to investigate whether it is feasible to obtain salivary cortisol levels in minipigs during performance of the standardized hoof trimming procedure and whether cortisol could serve a quantitative marker of stress and behavioral pathology in tgHD minipigs. We hypothesized that tgHD minipigs show altered stress response compared to wt minipigs and performed a baseline and follow-up assessment after one year to assess test-retest reliability and longitudinal behavior of the measure.

MATERIALS AND METHODS

Animals and housing conditions

The study was conducted with 32 adult, female Libechov minipigs, 14 tgHD (124 Q) and 18 wt controls. The Libechov minipig is a mixture of five different races, the Goettingen minipig, Minnesota minipig, Cornwall minipig, Large Black and Large White minipig [28]. The animals were bred at the Institute of Physiology and Genetics in Libechov, Czech Republic. With a life expectancy of around 12–15 years [29] and an adult body weight of around 46–128 kg [30] they have the propensity to serve as a large animal (LA) model with a high genetic homology compared to humans (approx. 98.5% ). All six groups were housed at the central animal facility (ZTE) of the University Hospital of Muenster (UKM), Germany, in unequally distributed mixed tg and wt groups in 12m² stables with a target temperature of 22°C and a humidity of 50–60% [21]. All stables were equipped with straw and hay as well as toys (e.g., balls and chains) for environmental enrichment. The pigs received a special feed two times a day (Wilhelm Reckhorn GmbH&Co. KG, 9091 Minipig Combi) and water ad libitum. A continuous medical surveillance was ensured by the ZTE. Resting values for the respiratory rate, body temperature, and the salivary cortisol (measured under familiar conditions inside the stable) were collected to provide a robust baseline (Table 1). Experiments were conducted in accordance and with permission of the local governmental animal protection agency prior to initiation of the study.

Table 1

Mean resting and stress values of (body temperature), respiratory rate and cortisol of the Libechov Minipig. Resting values were measured inside the stable, stress values were measured during the hoof trimming procedure (temperature, respiratory rate) and directly afterwards (cortisol)

	Resting Value (mean)	n	Stress Value (mean)	n
Temperature [°C]	###	###	38.67	58
Respiratory rate [1/min]	33.23	13	35.05	59
Cortisol [μg/dl]	0.341	62	0.896	57

Experimental setup

Between August 2014 and December 2016, every pig received two hoof trimming sessions with stress test assessments at baseline (visit 1 - V1) and after 1 year (visit 2 - V2) at the ZTE. During the V1 procedure, the animals were 2-3 years old and were well accustomed to their environment in the ZTE stables. The hoof trimming cage (HTC) was custom-made (Bilfinger GmbH, Germany) to fit the size of the animals and the needs of this project. Figure 1 shows pictures of the HTC. The cage is 130 cm long, 80 cm high, 40 cm wide and covered with stainless steel sheets (tare weight approximately 100 kg). Lockable lattice doors are available at each end. The base of the HTC contains a fixed, rounded and padded pole (8 cm diameter). The animals were placed one foreleg and one hind leg on each side of this pole when entering the cage and rested with their breast and abdomen on the pole. The cage was lifted up with a crane (Hadef GmbH, Germany) so the hoofs could be easily accessed approximately 30 cm above the ground from all sides. Four extendable support legs were used to provide a firm stand avoiding displacement of the cage. Although the side walls of the HTC were closed, direct visual contact with the minipig was available at both ends of the cage.

Fig.1

(A) The hoof trimming cage (HTC). HTC front face with lockable doors on each of the short sides. (B) Long side of the HTC; covered with stainless steel sheets. Minipig inside is lifted up with all hoofs accessible for hoof trimming. (C) Front face of the HTC with minipig inside during saliva sample collection.

Experimental setup: Procedure of hoof trimming and saliva collection

Salivary cortisol samples were collected before, during and after the hoof trimming procedures at the same time intervals V1 and V2 (Table 2). Pigs were allowed to chew on a piece of cotton until saliva was available for collection (samples a, b, c, d, e, and f). The respiratory frequency and body temperature were measured three times during hoof trimming (respiratory frequency = rf1, rf2, rf3; body temperature = t1, t2, t3). The first cortisol sample (sa; resting cortisol) was collected within the familiar environment of the stable to ascertain a minimal stress exposure. Afterwards, the pig left the stable, voluntarily entered the HTC and was rewarded with treats (Wilhelm Reckhorn GmbH & Co. KG, Altromin 9053 special diet for Minipigs). Once the minipig was inside the cage, all doors were closed. The pulse oximeter was fastened on the minipigs tail and the heart rate was documented every 30 seconds. The cage was lifted (crane up = cu) and 15 min after collection of the resting cortisol a second sample (sb) was collected and the first respiratory rate (rf1) and temperature (t1) were recorded. The length hoof trimming procedure (applying a gripper) followed (length correction front/hind right/left = lfr, lfl, lhr, lhl) and took about two minutes per hoof. This was followed by the third saliva collection (sc) and second measurement of respiratory rate (rf2) and temperature (t2). The angle hoof trimming (applying an angle grinder) (angle correction front/hind right/left = afr, afl, ahr, ahl) took an additional two minutes per hoof and was followed by the fourth saliva collection (sd) and third vital sign measurements (rf3, t3). Subsequently, the cage was lowered (crane down = cd) to the ground, the animal left the cage and re-entered the stable. Five and ten minutes after the fourth saliva sample, the fifth (se) and sixth (sf) samples were collected inside the familiar environment of the stable. The whole procedure lasted approximately 45 minutes; see Table 2 for a timeline.

Table 2

Standardized schedule of the stress test

Time [min.]	0	5	10	15	20	25	30	35	40	45
Location	stable	stable	HTC	HTC	HTC	HTC	HTC	HTC	stable	stable
Pulse			x	x	x	x	x	x
Saliva*	x			x		x		x	x	x
Respiratory rate	x			x		x		x
Temperature				x		x		x

*saliva samples a, b, c, d, e, f. whole procedure inside the HTC (= manipulations): cu = crane up, rf1 = respiratory frequency 1, t1 = body temperature 1, sb = saliva sample b, lfr = length correction front right, lfl = length correction front left, lhr = length correction hind right, lhl = length correction hind left, rf2 = respiratory frequency 2, t2 = body temperature 2, sc = saliva sample c, afr = angle correction front right, afl = angle correction front left, ahr = angle correction hind right, ahl = angle correction hind left, rf3 = respiratory frequency 3, t3 = body temperature 3, sd = saliva sample d, cd = crane down.

Measuring saliva samples via ELISA

The measurement of the saliva samples was performed using a competitive ELISA procedure (IBL International GmbH, Germany). All saliva samples collected in the ZTE were frozen overnight at – 20°C. The next day, the samples were defrosted at room temperature (RT) and centrifuged for 10 minutes at 2500×g to extract the saliva from the cotton. The supernatant was filled into labeled Eppendorf tubes and stored at – 20°C until further processing. On the day of measurement, the samples were defrosted and all reagents brought to room temperature (18–25°C). The ELISA was conducted as described in the IBL manual for cortisol ELISA (IBL International GmbH, Germany). At the end of the test the optical density of the ELISA was measured with a photometer (Thermo Scientific Multiskan FC, Vantaa, Finland) at 450 nm (reference-wavelength: 600–650 nm) within 15 minutes after adding the stop solution. For all assessments, quality controls were within the acceptable ranges as stated on the labels and the IBL QC certificate (IBL International GmbH, Germany).

Statistics

The statistical evaluation was carried out with RStudio (Version 3.1.1, R Foundation for Statistical Computing, Vienna, Austria, 2014). To analyze differences between tg and wt minipigs over the 45 minute assessment a 2 (group)×6 (cortisol sample time), a 2 (group)×3 (respiratory rate/temperature time point) and a 2 (group)×19 (manipulations) repeated measures ANOVA was conducted for V1 and V2, respectively. To analyze differences between tg and wt pigs over the 45 minute assessment a 2 (group)×time ANOVA was conducted for V1 and V2, respectively. The two groups (tg, wt) have been compared across the six saliva samples, three time points of respiratory rate/temperature measurements and 19 manipulations inside the HTC. Differences between tg and wt animals within the sample times were analyzed with the Wilcoxon-Mann-Whitney Test. This test was performed, if the ANOVA showed a (sample) time effect. In this case the difference between (sample) times across groups (n = 32) was tested for exploratory purposes. The significance level was set to p≤0.05 (*indicates p≤0.05, **indicates p≤0.01, ***indicates p≤0.001). No adjustment for multiple testing was performed.

RESULTS

Increased cortisol concentrations [μg/dl] were detected at V1 (Fig. 2A) and V2 (Fig. 2B) assessments and a comparison between the two visits (Fig. 2C). There is an overall sample time effect (n = 32) at both visits (p < 0.001). The V1 comparison between tgHD and wt minipigs (post-hoc analysis using the Wilcoxon-Mann-Whitney Test) shows a statistical trend suggesting a higher cortisol concentration in tg minipigs in the last two samples e (p = 0.09) and f (p = 0.08). Longitudinally a significant difference in the rise of the cortisol concentration was observed between the genotypes (ANOVA: p = 0.03). At V2 a significant difference (p < 0.05) was detected in the last sample f, with a higher cortisol measured in the wt animals but no difference between the genotypes across the whole assessment (ANOVA: p > 0.05). Figure 2C compares cortisol levels between V1 and V2 assessments. Next to the significant time effect reported in V1 and V2, there is also a visit effect (ANOVA: p = 0.03) with a higher cortisol in all animals (n = 32) during the V1 assessment.

Fig.2

Rise of the cortisol level [μg/dl] during the stress test in the visit 1 (V1) (A) and visit 2 (V2) assessment (B). (C) Comparison of the rise of cortisol in V1 and V2 with n = 32. Sample a indicates the resting cortisol inside the stable, samples b, c and d are collected during the hoof trimming and sample e and f after the trimming, back inside the stable. [box-and-whisker-plots with inter-quartile ranges; *indicates p≤0.05,+indicates p = 0.05–0.1].

Figure 3 shows the change in the respiratory rate during the hoof trimming procedure (time inside the cage) at V1 (Fig. 3A) and V2 (Fig. 3B) assessments and the comparison between the two visits (Fig. 3C). There was a time effect in the change of the respiratory rate at both visits (V1: p < 0.001, V2: p < 0.01), apparent as a decrease of the respiratory rate during the hoof trimming procedure. A post-hoc analysis using a Wilcoxon-Mann-Whitney Test (difference between every time point) showed a significant decrease from time point 1 to 3 of the respiratory rate in V1 (p < 0.001) and V2 (p < 0.01). There was no significant difference between the genotypes within every time point (Wilcoxon: p > 0.05) as well as longitudinally (ANOVA: p > 0.05). Figure 3C compares the change of the respiratory rate between V1 and V2 assessments and an ANOVA revealed a significant visit effect (ANOVA: p = 0.04) with a slightly higher respiratory rate during the V2 assessment.

Fig.3

Longitudinal change of the respiratory rate during the hoof trimming. The rate is measured at three different time points (1, 2 and 3) during the trimming, with around 10 minutes between every time point. (A) V1 assessment; (B) V2 assessment; (C) Comparison of the change of the respiratory rate in V1 and V2 with n = 32. [box-and-whisker-plots with inter-quartile ranges; *** indicates p≤0.001, **indicates p≤0.01].

Figure 4 shows the increase of body temperature during hoof trimming (time inside the cage). A time effect could be detected in the V1 and V2 assessment (Fig. 4A: p < 0.001 and B: p < 0.01). A Wilcoxon-Mann-Whitney Tests (tested between every time point 1–3) revealed significant increases between t1 and t3 (Fig. 4A and B: p < 0.01). There were no significant differences between the genotypes at each of the three time points (Wilcoxon: p > 0.05), however, time point 1 in V2 exhibited a trend (p = 0.06) for a higher temperature in wt animals. In addition, the longitudinal analysis shows no difference between tg and wt animals at V1 and V2 (ANOVA: p > 0.05). Figure 4C compares the overall change of the temperature between V1 and V2 assessments. Next to the significant time effect in both visits there was a significant visit effect (ANOVA: p < 0.001) with a higher temperature observed overall during V1, while post-hoc Wilcoxon tests at each visit were not significant.

Fig.4

Change of the temperature (t) [°C] rate during the hoof trimming. The temperature is measured at three different time points (1, 2 and 3) during the trimming, with around 10 minutes between every time point. (A) V1 assessment; (B) V2 assessment; (C) Comparison of the change in temperature in V1 and V2 with n = 32. [box-and-whisker-plots with inter-quartile ranges; **indicates p≤0.01].

Figure 5 shows the minimum, maximum and mean pulse rates of the tg and wt minipigs during the whole stress test in the V1 (Fig. 5A) and V2 (Fig. 5B) assessments. During V1, a significant difference was seen in the minimum pulse rate between the two genotypes (Wilcoxon: p < 0.05) with the wt animals exhibiting a higher minimum pulse. No differences between the genotypes could be detected during V2.

Fig.5

Minimum, maximum, mean and median pulse during the assessment divided by genotype. (A) V1 assessment; (B) V2 assessment. (tg = transgenic, wt = wildtype) [box-and-whisker-plots with inter-quartile ranges; *indicates p≤0.05].

The alteration of the pulse rate in response to the manipulation (= assessment time) is shown at V1 (Fig. 6A, D) and V2 (Fig. 6B, E). In V1 a time effect (p < 0.001) was seen during the assessment (from the first, “crane up” [cu], to the last, “crane down” [cd], manipulation), represented by decreasing pulse rates across the different manipulations. There also was a genotype effect across the assessment time (ANOVA: p = 0.03) with wt animals having a significantly higher pulse in the first (cu) and last (rf3, t3, s4 and cd) manipulations (Wilcoxon: p < 0.05, p < 0.01). This longitudinal genotype effect is highlighted in Fig. 6D. The V2 assessment showed a time affect, too (p < 0.001). However, the longitudinal difference between wt and tg animals only showed a trend at V2 (ANOVA, p = 0.09) (Fig. 6E). The within group analysis (Wilcoxon) showed a significantly higher pulse in the wt minipigs during the “t1” and “ahr” manipulations (Fig. 6B, p < 0.05). Figure 6C shows a comparative illustration of V1 and V2 with a significant visit effect (p < 0.001), i.e., a higher pulse rate at the V1 assessment.

Fig.6

Change of the pulse rate as a function of/according to the manipulation during the hoof trimming (time inside the cage) while V1 (A and D) and V2 (B and E) assessment. (A and B) Change of the pulse rate divided by genotype; (C) Comparison between V1 and V2 pulse during manipulation; (D and E) line graphs of the genotypes. [cu = cage up, rf1 = respiratory frequency no.1, t1 = temperature no.1, sb = saliva sample b, lfr = length correction front right, lfl = length correction front left, lhr = length correction hind limbs right, lhl = length correction hind limbs left, rf2 = respiratory frequency no.2, t2 = temperature no.2, sc = saliva sample c, afr = angle correction front right, afl = angle correction front left, ahr = angle correction hind limbs right, ahl = angle correction hind limbs left, rf3 = respiratory frequency no.3, t3 = temperature no.3, sd = saliva sample d, cd = crane down] [**indicates p≤0.01, *indicates p≤0.05].

DISCUSSION

This study showed that conducting the stress test assessing salivary cortisol concentrations was feasible and well tolerated in tgHD and wt minipigs. The test was applied repeatedly without relevant side effects. Cortisol levels were sufficiently reproducible and showed acceptable variability. The study provided reference values for cortisol levels at rest and under stress exposure in minipigs.

Cortisol concentration exhibited slight genotype differences within the last saliva sample (f) of the V2 assessment only, with wt animals exhibiting a higher cortisol level compared to tgHD minipigs. However, the genotype changes observed were marginal and the biological significance of this observation is unclear. Interestingly, as mentioned in the introduction, one human study suggested that early-HD might be associated with hypocortisolism [19]. However, other studies found increased cortisol levels in HD gene carriers [17, 18].

These results suggest variability in the characteristics of cortisol metabolism across species, cohorts and studies. Whether in HD patients this could be explained by different severity of clinical psychiatric features, which are known to be variable as they are influenced by symptomatic therapy [31, 32], remains to be elucidated. We recently learned that tgHD minipigs at the ages investigated in this study do not yet exhibit measurable motor, cognitive or behavioral deficits [21, 33]. Therefore, similarly to those domains, manifestation of changes in cortisol metabolism of tgHD minipigs may require older animals, i.e., longer observation periods. Thus, a longer study may be warranted to re-assess a possible cortisol genotype association.

Circadian changes in cortisol levels may also induce relevant variability. Salivary same as systemic cortisol profiles show a typical diurnal profile with highest concentrations seen in the morning shortly after awaking in humans [34], chimpanzees [35], and pigs [36 –38]. Also studies in R6/2 mice showed a dysregulation of the circadian rhythmicity of corticosterone over a 24 h period, which supported an abnormal HPA-axis activity [9]. HPA-axis hyperactivity was reported in HD patients with higher basal cortisol levels in the morning compared to controls [18, 39]. Salivary cortisol showed an altered cortisol awakening response about 45–60 min post-awakening even in pre-manifest HD patients prior to the onset of motor symptoms [13]. Therefore, future studies should explore and control for the impact of circadian changes in cortisol levels of minipigs.

Interestingly, higher cortisol levels were seen across genotypes during the initial V1 assessment compared to the follow-up V2 visit. This may suggest a habituation effect, as minipigs may remember the HTC and procedure of hoof trimming and therefore exhibit a reduced response to the stressor at re-exposure. We previously reported evidence for good cognitive performance of minipigs in other tests [21, 33], which may support this assumption.

As expected the resting cortisol levels (sample a) were not significantly different at both visits. These samples were collected inside the stables and should not be influenced by the HTC or hoof trimming procedure. This observation supports the reliability and reproducibility of the measures obtained.

Importantly, a reliable increase of cortisol was observed around 30–45 min after collecting the resting cortisol samples (sample a) inside the stable. We therefore conclude that the hoof trimming paradigm as such provided an appropriate stressor to trigger a measurable and reproducible cortisol response. As mentioned before, other studies demonstrated similar increase around 30 min after stress exposure in different animal models [16 , 23–25]. Therefore, our results are consistent and extend the available observations across species to minipigs.

The respiratory rate decreased over time, whereas the temperature increased between the first and last assessment. The decreased respiratory rate might be associated with the inability to move inside the HTC. With regards to the increase in body temperature, other studies demonstrated, that this often accompanies psychological stress [40, 41]. The comparison of the two visits showed a significant visit effect in both, respiratory rate and temperature. Body temperatures changed in parallel to cortisol with higher values at the first visit. However, the respiratory rate suggested an inverted effect, with a higher rate at V2. The biological significance of this observation is uncertain and this needs to be reassessed in future studies.

The minimum pulse rate of the minipigs showed a significant difference between the two genotypes in the V1 assessment. During this time, wt animals had a higher pulse than the tg animals. As this effect could not be confirmed in visit 2, the biological relevance is uncertain. With regards to effects of stressors on the pulse rate, a time and genotype effect is visible in both visits. Within the V1 and V2 assessments, both groups decreased in their pulse rate. The high start pulse might be due to the fact the animals were excited when they entered the cage for the first time and were lifted up. Afterwards the pulse rate decreased slowly with the wt animals having a significantly higher pulse at the end of the hoof trimming in the V1 and a trend for a higher pulse in the V2 assessment.

In summary, the comparisons of the two visits suggest that stress responses in the minipigs were higher during the V1 assessment when they were exposed to the setup and procedure for the first time.

We conclude that the stress test described induced relevant stress in the animals resulting in a consecutive and well detectable increase in cortisol concentrations, which can be measured non-invasively in saliva samples. The stress test is a safe procedure [42] and compliant with animal welfare requirements. A better understanding of the HPA-axis may help to develop therapies targeted to improve the management of stress responses in HD patients [5]. As there is still no cure for HD patients, effective strategies to manage symptoms remain of great interest. With cortisol as a reproducible marker for stress and behavioral burden in minipigs, assessment of behavioral aspects may be facilitated in future preclinical studies using minipig models of HD.

FINANCIAL DISCLOSURE

The George-Huntington-Institute has received grant support from the CHDI Foundation to conduct the work reported in this study. The authors declare no other financial interests related to this manuscript.

CONFLICT OF INTEREST

The authors have no conflict of interest to report.

Footnotes

ACKNOWLEDGMENTS

This study was funded by the CHDI foundation (). Parts of this study were supported by donations from private donors interested to support research in HD. We thank the team of the Zentrale Tierexpermientelle Einrichtung (ZTE) of the University Hospital of Muenster, Muenster, Germany, for continuous support.

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