BRIEF RESEARCH REPORT published: 20 July 2021 doi: 10.3389/fnagi.2021.683412 Preserved Thermal Pain in 3xTg-AD Mice With Increased Sensory-Discriminative Pain Sensitivity in Females but Affective-Emotional Dimension in Males as Early Sex-Specific AD-Phenotype Biomarkers Toni Cañete 1,2 * and Lydia Giménez-Llort 1,2 * 1 Department of Psychiatry and Forensic Medicine, Autonomous University of Barcelona, Barcelona, Spain, 2 Institut de Neurociències, Autonomous University of Barcelona, Barcelona, Spain Edited by: Lakshmi Rajagopal, Northwestern University, The increase of the aging population, where quite chronic comorbid conditions are United States associated with pain, draws growing interest across its investigation and the underlying Reviewed by: nociceptive mechanisms. Burn injuries associated problems might be of relevance in Chase Matthew Carver, The University of Texas Health the older adult’s daily life, but in people with dementia, exposure to high temperatures Science Center at San Antonio, and heat sources poses a significantly increased risk of burns. In this brief report, the United States Hudson Sousa Buck, hind paws and tail pain withdrawal reflexes and the emotional responses to thermal Santa Casa of Sao Paulo, Brazil nociception in 3xTg-AD mice were characterized for the first time in the plantar test *Correspondence: and compared to their non-transgenic (NTg) counterparts. We studied a cohort of Toni Cañete male and female 3xTg-AD mice at asymptomatic (2 months), early (6 months), middle antoni.canete@uab.cat Lydia Giménez-Llort (9 months), and advanced (12 and 15 months) stages of the disease and as compared lidia.gimenez@uab.cat to sex- and age-matched NTg control mice with normal aging. At 20 and 40W intensities, the sensorial-discriminative thresholds eliciting the withdrawal responses Received: 20 March 2021 Accepted: 25 May 2021 were preserved from asymptomatic to advanced stages of the disease compared to Published: 20 July 2021 NTg counterparts. Moreover, 3xTg-AD females consistently showed a greater sensory- Citation: discriminative sensitivity already at premorbid ages, whereas increased emotionality was Cañete T and Giménez-Llort L shown in males. False-negative results were found in “blind to sex and age” analysis, (2021) Preserved Thermal Pain in 3xTg-AD Mice With Increased warning about the need to study sexes independently. The current results and previous Sensory-Discriminative Pain report in cold thermal stimulation provide two paradigms unveiling sex-specific early Sensitivity in Females but Affective-Emotional Dimension in AD-phenotype nociceptive biomarkers to study the mechanistic underpinnings of sex-, Males as Early Sex-Specific age- and AD-disease-dependent thermal pain sensitivity. AD-Phenotype Biomarkers. Front. Aging Neurosci. 13:683412. Keywords: pain, plantar test, biomarkers, nociception, sensory-discriminative, affective-emotional, translational doi: 10.3389/fnagi.2021.683412 research, gender medicine Frontiers in Aging Neuroscience | www.frontiersin.org 1 July 2021 | Volume 13 | Article 683412 Cañete and Giménez-Llort Sex-Dependent Thermal Pain in AD INTRODUCTION yield some insights about the aversive or emotional aspect of pain (Mogil and Bailey, 2010). Chronic pain is one of the most frequent causes of suffering and The burden of pain is greater, more varied, and more variable disability, affecting the individual’s quality of life. The increase for women than men (Cravello et al., 2019). Sex and gender of the aging population, where quite comorbid conditions are differences in nociception and pain in aging are an important associated with chronic pain, draws growing interest across field of study. The understanding of these differences relays in its investigation (Domenichiello and Ramsden, 2019). The biological (genetics and sex hormones), psychological (negative neural processes of perceiving, encoding, and processing noxious emotions, coping strategies), lifestyle, and socio-cultural factors stimuli (nociception) as well as the unpleasant sensory and that change across each individual’s life span (Berkley et al., emotional experience associated with actual or potential tissue 2006; Keogh, 2006). For at least the past decade, sex and age damage (pain) define the two inseparable levels of study of differences have been explored in some detail in rodent models one of the most relevant phenomena preserved until the (Giménez-Llort and Pick, 2017). However, this brief research very last seconds of life, becoming an important field of report was aimed to characterize the sensory-discriminative and both neuroscience and medical research (Loeser and Treede, emotional responses to plantar and tail nociceptive thermal 2008). In addition, burn injuries’ sensitivity-related problems (heat) stimulation in male and female 3xTg-AD mice harboring might be of relevance in the daily life of the older person. PS1M146V , AppSwe , tauP301L transgenes (Oddo et al., 2003b) Older adults are one of the more susceptible populations to as compared to Non-transgenic (NTg) counterparts with the suffering burn injuries due to thinning skin and the aging same genetic background (C57BL/6J×129/Sv). The novelty of process involving a progressive decline of sensory function studying pain responses in the 3xTg-AD model, at a spectrum (Jeschke and Peck, 2017). In people with dementia, exposure of ages from young adulthood to old age, is that in the to high temperatures and heat sources poses a significantly 3xTg-AD mice these ages mimic asymptomatic to advanced increased risk of burns (Harvey et al., 2016). The neural neuropathological stages of AD. In fact, this animal model processes of encoding and processing noxious stimuli differ of Alzheimer’s disease progressively develops βA plaques and from normal aging in these people. The sensory-discriminative neurofibrillary tangles with a temporal-and regional- specific dimension is maintained, increased pain tolerance is yet profile that closely mimics their development in the human unclear, but the cognitive-evaluative and affective-emotional AD brain (Barrot, 2012). Synaptic dysfunction manifests in dimensions seem to be affected. During the progress of an age-dependent manner starting at 6 months of age but Alzheimer’s disease (AD) and other dementias, a difficulty before plaque (12 months of age) and tangle (15 months of verbal expression ending in aphasia is added to the of age) hallmark neuropathology (Belfiore et al., 2019) and increasing severity of cognitive problems, with the consequent severe cognitive impairment (Chapman et al., 1985; Giménez- worsening or impossibility of trustful self-report. All these factors Llort et al., 2007; Baeta-Corral et al., 2015). 3xTg-AD mice contribute to pain being under-detected and under-treated, show diminished curiosity and apathy (exploration) in the a critical scenario still demanding important research efforts open-field and lack of ability to cope with mild stressors (novelty) (Giménez-Llort et al., 2020). with increased emotionality (freezing behavior, defecation, and Both in the clinical and basic research scenarios, the urination) in most of the behavioral tests (Oddo et al., 2003a,b; impact of pain in cognition has been largely studied, but how Billings et al., 2005; Giménez-Llort et al., 2007). At early nociception and pain sensitivity are modified in individuals and advanced stages of the disease, male 3xTg-AD show with several cognitive impairment levels is a research field preserved tail flicking withdrawal response to a cold stimulus and that still needs more endeavors (Defrin et al., 2015). At the presented a correlation with the immediate response exhibited translational level, pain studies rely extensively on animal in a sort of environments differing in the anxiogenic levels models to assess this process’s sensory and psychological (Baeta-Corral et al., 2015). complexities, and rodents are employed in the majority of this research (Giménez-Llort and Pick, 2017). Due to ethical MATERIALS AND METHODS considerations, research paradigms involving noxious stimuli elude those eliciting vocal expression of pain and relay in Subjects others based on withdrawal responses to avoid the stimulus, In a cohort of a total of 216 animals of our 3xTg-AD with a translation to those seen and assessed in humans and NTg mice colonies established at the Behavioral Facility through behavioral scales. For the investigation of thermal Core, Universitat Autònoma de Barcelona were used (Oddo nociception in mice, the hot plate test’s withdrawal response et al., 2003b). Two to four littermates of the same genotype, requires both a supraspinal and a spinal mechanism. In contrast, age and sex were grown and maintained in macrolon cages a spinal reflex mediates the elicitation of the tail-flick and (35 cm × 35 cm × 25 cm) under standard laboratory conditions hind paws-withdrawal response following exposure to a heat of food and water ad libitum, in a temperature-controlled room stimulus in the radiant heat test (Plantar test or Hargreaves’s 22 ± 2◦ C, a 12 h light/dark cycle (lights on at 8:00 a.m.) and test), although supraspinal mechanisms can modulate this reflex relative humidity of 50–60%. A 2 × 2 × 5 experimental design (Chapman et al., 1985; Barrot, 2012). Pain discrimination included 55 male and 53 female 3xTg-AD mice at asymptomatic measures in the radiant heat assay provide information about (2 months of age), early (6 months of age), middle (9 months the pain experience’s sensory-discriminative aspect, yet they can of age), advanced βA (12 months of age) and tau (15 months Frontiers in Aging Neuroscience | www.frontiersin.org 2 July 2021 | Volume 13 | Article 683412 Cañete and Giménez-Llort Sex-Dependent Thermal Pain in AD of age) pathology stages of the disease that were evaluated to either 20% (Low-20W) or 40% (High-40W) active intensity and also compared to sex- and age-matched NTg control mice halogen bulb. These heat stimuli were applied underneath the (52 males and 56 females). Animals were tested during the light plantar surface of the mice’s hind paws (both right and left) phase, between 09:00 a.m. and 02:00 p.m., and following Spanish and tail, through the base of the plastic box, every 10 min. legislation on ‘‘Protection of Animals Used for Experimental and Three alternate measures of withdrawal latency were established Other Scientific Purpose’’ and EU council directive (2010/63/UE) at 2 min-intervals in each paw, with a minimum of 2 s and on this subject. a maximum of 15 s (cut-off latency). Paw and tail withdrawal latencies were recorded, and the three determinations were Plantar Test—Hargreaves Test averaged for each animal. A cut-off latency of 15 s was imposed Thermal sensitivity to noxious heat was tested using a Plantar test to avoid tissue damage. The affective-emotional dimension was (Ugo Basile, Italy; Hargreaves et al., 1988) with a modification measured as the total number of defecations, a classical measure of the protocol to Cheah et al. (2017). Mice were habituated of the animals’ emotionality, recorded until the end of the test to the experimental room for at least 60 min before behavioral (see Figure 1). assessment. Following habituation, the animals were placed in a clear plastic chamber (a transparent Plexiglas box, 7.0 × 12.5 cm and 17.0 cm high) and left to acclimatize for 5 min before Statistics testing (habituate the animals to the testing chamber). During Statistical package for social science was used (SPSS, version this time, the mice initially demonstrated exploratory behavior 25.0 software). Data are expressed as mean ± SEM. Effects of but subsequently stopped exploring and stood quietly with factors genotype (G), sex (S), and age (A) in a 2 × 2 × 5 factorial occasional grooming bouts. design were analyzed with ANOVA followed by Post hoc The sensitive-discriminative dimension of thermal pain was Duncan’s multiple range test. In sex and age blind analysis, measured as follows: A mobile radiant heat source (infrared light Student’s t-test was used. A probability value of less than 0.05 stimulus), which was located 0.5 cm under the glass floor, was set (p < 0.05) was considered statistically significant. FIGURE 1 | Schematic representation of the plantar test of thermal stimuli protocol used. Sensory-discriminative dimension of thermal withdrawal response in male and female 3xTg-AD and age-matched NTg mice with normal aging. (A) At low-20W and high-40W active intensity, infrared light stimuli were applied underneath the plantar surface of the mice’s hind paws (both right and left) and tail every 10 min. Paw (PWL) and tail (TWL) withdrawal latency were recorded for a sensory-discriminative dimension and the total number of defecations for an affective-emotional dimension. (B) Hind paw withdrawal responses at low-intensity 20-W (C) and high-intensity 40-W (D). Tail withdrawal at low-intensity 20-W (E) and high-intensity 40-W. Groups: NTg, non-transgenic mice (open bar); 3xTg-AD, transgenic mice (male “black,” female “red” bar). Error bars represent mean ± SEM. Statistics: Sex effect, S***, p < 0.001 or S** p < 0.01. Student t-test comparisons are shown in the graphs as # p < 0.05 vs. the NTg-group. Frontiers in Aging Neuroscience | www.frontiersin.org 3 July 2021 | Volume 13 | Article 683412 Cañete and Giménez-Llort Sex-Dependent Thermal Pain in AD FIGURE 2 | Affective-emotional dimension associated with thermal withdrawal response in male and female 3xTg-AD and sex- and age-matched NTg mice with normal aging. Total number of defecations. Groups: NTg, non-transgenic mice (open bar); 3xTg-AD, transgenic mice (male “black,” female “red” bar). Error bars represent mean ± SEM. Statistics: Student t-test comparisons are shown in the graphs as # p < 0.01 vs. the NTg-group. RESULTS To avoid errors in the measurement and confounding factors that may elicit a higher sensitivity and response to thermal nociception (Barrot, 2012), it is important to bear in mind that different arousal and attention states, such as alert, resting, and light sleep, could be important modulators of pain sensitivity. Besides, it is important to avoid the testing while the subject is grooming (Callahan et al., 2008). Therefore, in this work, animals were acclimated to the testing apparatus. This procedure decreases the level of stress in the animal, its activity, and the variability in the data while not affecting the mean response times. As summarized in Figure 1A, measures were done in the left and right hind paws and tail of habituated animals using two intensities of the infrared light stimulus (20W and 40W). Figures 1B–E and 2 depict the results per sex and age of the sensory-discriminative dimension of the thermal withdrawal response and the associated affective-emotional dimension, respectively. Figure 3 provides the global genotype analysis of FIGURE 3 | Genotype effects in the Sensory-discriminative and these results in a sex and age blind analysis (pooled). affective-emotional dimensions blind to sex and age in 3xTg-AD and NTg Age effects were found in hind paw withdrawal response mice with normal aging. Represented by hind paw withdrawal responses at low (20-W) and high intensity (40-W), Tail withdrawal at low (20-W) and high latencies in both intensities (20-W; A, F (4,215) = 2.49, intensity (40-W) and total defecations. Groups: NTg, non-transgenic mice p = 0.044 and 40-W; A, F (4,215) = 2.56, p = 0.040), showing (open bar); 3xTg-AD, transgenic mice (green bar). Error bars represent higher latency at 6 months of age compared to other ages. mean ± SEM. Statistics: Student t-test comparisons are shown in the graphs Tail withdrawal response also presented higher latencies at as # p < 0.01 vs. the NTg-group. 2 and 6 months of age than at the rest of the ages (40-W; F (4,215) = 8.98, p < 0.001). Preserved thermal pain withdrawal responses in 3xTg-AD all ages, Paw-20W; #G (genotype effect), t (107) = 4.30, mice were found in both sexes during asymptomatic stages p < 0.001. Paw-40W; #G, t (107) = 2.42, p = 0.0017. Tail- of the disease. Besides, in the case of females, 3xTg-AD mice 20W; G, t (107) = 3.39, p = 0.001. Tail-40W; G, t (107) = 3.43, exhibited shorter hind paw and tail withdrawal latencies p = 0.001), indicating increased sensory-discriminative than controls at the two intensities studied (Figures 1B–E, pain sensitivity. Frontiers in Aging Neuroscience | www.frontiersin.org 4 July 2021 | Volume 13 | Article 683412 Cañete and Giménez-Llort Sex-Dependent Thermal Pain in AD Sex effects were found in hind paw withdrawal response the 3xTg-AD mice (F (1,215) = 10.42, p = 0.001) compared latencies, in both low and high intensities (Figures 1B,C, to controls. 20-W; S∗∗∗ , F (1,215) = 33.46, p < 0.001 and 40-W; S∗∗∗ , F (1,215) = 31.63, p < 0.001) and the tail latency at 40-W DISCUSSION (Figure 1E, S**, F (1,215) = 10.09, p = 0.002). Further analysis per sex at each age indicated faster hind paw (20-W, #G, Nociceptive pain is a function of the nervous system that at 2, 6, and 9 months of age, t (12) = 2.38, p = 0.035, provides important sensory information about our environment t (12) = 2.55, p = 0.025 and t (23) = 2.55, p = 0.018 respectively; and external agents, being a protective mechanism for tissue 40-W, #G, at 9 months of age, t (23) = 4.42, p < 0.001) damage. While special interest groups join efforts to improve and tail (20-W, #G, at 2, 9, and 15 months of age, pain assessment and intervention in older people with dementia t (12) = 2.12, p = 0.045, t (23) = 2.15, p = 0.042 and is growing (Defrin et al., 2015; Domenichiello and Ramsden, t (27) = 1.43, p = 0.001 respectively and 40-W, #G, at 6, and 2019). 15 months of age, t (12) = 3.17, p = 0.008 and t (27) = 0.17, The main findings of the present study provided evidence p < 0.001) withdrawal responses of female 3xTg-AD mice that, under both 20W and 40W intensities studied, the sensory- as compared to controls. Also, in females, these genotype discriminative threshold to a thermal stimulus in this cohort effects were already shown at asymptomatic ages (2 months of male and female 3xTg-AD mice was preserved from the of age) as unveiled by low intensity (hind paw, t (12) = 2.38, asymptomatic (2 months of age) to advanced stages of the disease p = 0.035 and tail t (12) = 2.12, p = 0.045). At high intensity, (15 months of age) as compared to sex- and age-matched NTg shorter latencies were found at early (tail, 6 months of mice with normal aging. Females exhibited increased sensory- age, t (12) = 3.17, p = 0.008) but also advanced (hind paw, discriminative pain sensitivity in both hind paw and tail, thus 9 months of age, t (23) = 4.42, p < 0.001; tail, 15 months indicating spinal and supraspinal mechanisms’ involvement. In of age, t (27) = 0.17, p < 0.001) stages of the disease, contrast, genotype differences in the emotional response were respectively (Figures 1B–E). better shown in males. The results blind to sex and age elicited Male 3xTg-AD only showed genotype effects in tail false negatives in the intensity of 40-W as the genotype effect was withdrawal response latencies, at early (20-W, #G, 6 months of lost when pooling the sex and age groups. age, t (10) = 2.57, p = 0.028 and 40-W, #G, at 6 months of age The present results showing preservation of pain sensitivity t (10) = 2.54, p = 0.029) and advanced (40-W, #G, at 12 months to a hot stimulus through aging and AD processes complement of age t (28) = 2.33, p = 0.027) stages of the disease. those previously reported by our laboratory in the tail-flick In summary, both low and high intensities, the withdrawal test (Baeta-Corral et al., 2015). The nociceptive response to responses in hind paws (spinal reflex) and tail (spinal reflex a cold stimulus was studied in male 3xTg-AD mice from subjected to supraspinal influences) consistently unveiled sex- early to advanced stages of disease (7 and 11 months of and genotype-dependent effects. However, 3xTg-AD female age, respectively) and found preserved. Tail-flick latency was mice consistently displayed shorter response latency to thermal only dependent on the age factor, and genotype effects were stimulation than female NTg mice, already at the low-intensity, shown as a loss of functional correlation between the different before advanced stages of disease (6- and 9-month-old) and at behavioral components of the AD-phenotype. In the present asymptomatic ages (2-month-old). Thus, the reduced thermal work with the opposed thermal stimulus, we studied the pain thresholds suggesting greater pain sensitivity were the plantar test response because it is a harmless and simple hallmark of the withdrawal response of 3xTg-AD females as nociceptive reflex. This choice was determinant since in the compared with their NTg counterparts. hot-plate test female C57BL/6 mice show preserved thermal Concerning the affective-emotional dimension, females and threshold at 3, 8, 15, and 28 months of age (Fahlström et al., males exhibited increased defecatory behavior as compared to 2011), but pain thresholds of female 3xTg-AD mice between their respective age and sex-matched controls at early (female 12 and 14 months of age did not differ from controls (Filali at 2 months of age, t (12) = −3.85, p = 0.002) and advanced et al., 2012). Other studies indicate that wild-type mice’s (male at 15 months of age, t (23) = −3.82, p = 0.001) stages pain sensitivity and emotional behavior increase with age, of the disease, respectively. However, the overall emotional showing an important age-dependent effect on the hot-plate response at all ages only reached statistical significance in males test. Instead, KO mice with a deletion of the p66 gene (t (105) = −3.60, p < 0.001). showed reduced pain sensitivity and emotionality (Berry et al., When results were studied blind to sex and age (Figure 3), 2007). In the hot-plate, the TASTPM mouse model of AD false-negatives appeared since genotype effects were only exhibited reduced thermal sensitivity to acute noxious heat detected in hind paw withdrawal response latencies at but also an increased opioidergic tone model (Aman et al., low intensity (20W; F (1,215) = 10.23, p = 0.002). The tail’s 2016). That study suggested increased inhibition/decreased withdrawal response was sensitive to genotypes at both excitation in the spinal cord as responsible for the reduced intensities (20-W and 40-W; F (1,215) = 5.93, p = 0.016 and thermal sensitivity associated with AD-related pathology in F (1,215) = 7.82, p = 0.006), always with a shorter latency this model. Similar studies in mutant mice with genetically- in the 3xTg-AD animals as compared to controls. induced hypoglutamatergic tone, showed an hyposensitive Regarding the emotional response, genotype differences phenotype to mechanical and thermal nociceptive stimuli, were shown as an increased number of defecations in thus supporting the idea that glutamate plays a key role Frontiers in Aging Neuroscience | www.frontiersin.org 5 July 2021 | Volume 13 | Article 683412 Cañete and Giménez-Llort Sex-Dependent Thermal Pain in AD in physiological nociception both in acute and chronic pain rats, along with increased plasma corticosterone concentrations (Kayser et al., 2015). (O’Mahony et al., 2009). The present work supports the research about the basic Anxiety and depression are related to pain, but their sex differences in pain, with considerable implications for the impact on emotionality may differ for males and females. clinic and how being blind to sex and age may lead to a false- In rats, it has also been observed that response to pain negative. Some studies amply demonstrate the hypothesis may determine other emotional behavior patterns, probably on fundamental neural, hormonal, and developmental reflecting different activation thresholds of some brain structures factors that mediate these sex/gender differences, which controlling anxiety. They found that high-sensitivity male reinforces the conclusion that no single answer can explain rats responded to an acute painful stimulation and showed sex/gender differences (Berkley et al., 2006). Currently, it significantly more freezing behavior. This suggests that animals is a clinical reality that women make up the large majority that are more vulnerable to stress might have innate deficits in of chronic pain patients (perhaps 70%). The involvement brain systems’ activity controlling the hypothalamic-pituitary- of individual genes in pain-related traits—demonstrated via adrenal axis that would typically allow them to cope with transgenic knockout mouse phenotypes or via quantitative stressful situations (Lehner et al., 2006). Female rats with trait locus (QTL; i.e., linkage) mapping—is sex-dependent maternal separation (a stressful process) had significantly on many occasions (Mogil and Bailey, 2010). Work in the longer paw lick latencies on a hot plate than handled rats, laboratory animal examined screening for pain phenotypes showing a persistent reduction in pain sensitivity. Therefore, and showed that C57BL/6 female mice were more sensitive the nociceptive circuitry involved in the hot plate’s responses than male mice, and similar sex differences were seen when appears particularly sensitive to early life stress (Weaver et al., analyzing the B6.MOLF12 congenic strain. The authors 2007). Since our first reports (Giménez-Llort et al., 2006), proposed that potential sex genotype interactions suggest 3xTg-AD mice show sex and age-dependent emotional and sex-specific genetic linkages (Mogil et al., 2006). A review BPSD-like derangements, with neophobia fear-related decrease provides evidence of sex differences in pain sensitivity and of rearing as the earliest behavioral marker emerging since the response to analgesic drugs in animals and humans. early premorbid-like stages of disease (Giménez-Llort et al., This summarizes that research on transgenic mice suggests 2007). For almost all behaviors assessed, male mice appear that normal males have a higher level of activity in the more fearful or deranged under fearful experimental conditions endogenous analgesic system than normal females. It is than females. The biological basis for these BPSD-like patterns postulated that males and females may have different was correlated to intraneuronal βA immunoreactivity in the pain modulatory circuits to suit these needs. Also, human basolateral amygdala (España et al., 2010). study has found that µ-opioid receptors in the healthy Glucocorticoid levels have been inversely correlated with female brain are activated differently from those in the risk-taking behaviors and found strongly increased in young healthy male brain (Kest et al., 2000). The response to adult females compared to age-matched males. Although k-opioids mediated by the melanocortin-1 receptor gene corticosterone was not measured in the present work, we in both mice and humans is also different for each sex have previously reported the sexual dimorphism in HPA axis (Wiesenfeld-Hallin, 2005). activity in these NTg mice, as measured by higher plasmatic Pain discrimination measures provide information about corticosterone levels in females than males at 6 months of the pain experience’s sensory-discriminative aspect but can age, and its strong reduction in old animals, due to an yield little insight into the aversive aspect of pain or the age-related decrease only observed in 15-month-old females but affective-emotional dimension. The current finding of increased not male (Giménez-Llort et al., 2008). In contrast, like in the defecation in the 3xTg-AD mice genotype and mainly greater AD-human patients (Rasmuson et al., 2002), the corticosterone in 3xTg-AD males than the NTg group indicates a higher levels of 15-month-old 3xTg-AD mice were higher than in level of emotionality. In previous studies, increased emotional age-matched NTg mice, but sexual dimorphism was lost due to behavior was also described from early to advanced stages of the increase of basal levels in males (Giménez-Llort et al., 2008). disease when 3xTg-AD mice confronted anxiogenic conditions, Furthermore, age-dependent sexual dimorphism in cognition from mild neophobia in the corner test or corridors resembling and stress response in the 3xTg-AD mice has also been reported burrows in the T-maze to the classical open and illuminated by other laboratories (Clinton et al., 2007). Therefore, the field test, among others (Giménez-Llort et al., 2008, 2010, 2007). plasmatic corticosterone pattern would agree with the patterns of Despite being a controversial issue about rodent validity and emotionality recorded here: increased emotionality in 2-month- reliability of defecation as an index of fear, there is sufficient old 3xTg-AD females but 15-month-old males. evidence using a large sample of animals (Aguilar et al., 2003). Finally, the most recent report in humans shows that In support of the pattern of sex effects reported here, about cognitively healthy apolipoprotein E4 (APOE4) carriers, with a the male as being more fearful than females, on the one demonstrated increased risk of late-onset AD, displayed lower hand, confirms Gray’s view (Gray and Buffery, 1971), even overall pain sensitivity non-carriers but greater unpleasantness a particularly relevant study with inbred Roman rats showed to thermal pain stimuli. This association of APOE4 allele status males defecated more than females in several different behavioral with an altered response to pain in a cognitively healthy sample tests (Aguilar et al., 2003). On the other hand, it is known of adults suggests that thermal evoked pain testing could serve that a pattern of increased fecal bolis is found in stressed as a potential phenotypic biomarker of individuals at increased Frontiers in Aging Neuroscience | www.frontiersin.org 6 July 2021 | Volume 13 | Article 683412 Cañete and Giménez-Llort Sex-Dependent Thermal Pain in AD risk for AD (Romano et al., 2021). The present work on the underpinnings of sex- and AD-disease-dependent modulation preserved withdrawal response in the 3xTg-AD mice since young of cold (Baeta-Corral et al., 2015) and hot (present work) age mimicks asymptomatic stages of disease agrees with this thermal pain sensitivity and be useful to progress in preclinical clinical finding and proposes sex-specific phenotypic biomarkers studies on pain management in dementia under a gender sensory-discriminative dimension in the female sex and affective- medicine perspective, with the aim of developing sex-specific emotional in males. analgesic therapies. Because the thermal pain measures were simply flat 20W and 40W intensity measures, the study has the limitation of DATA AVAILABILITY STATEMENT not fully describing threshold intensity of limb/tail withdrawal. The study of only a mild thermal pain model may also seem The raw data supporting the conclusions of this article will be one-dimensional to cover sensory-discriminative responses. made available by the authors, without undue reservation. Therefore, future studies may better elucidate incremental pain threshold response. On the other hand, in order to reduce to ETHICS STATEMENT minimum confounding factors a cohort of animals of different ages, grown and aged under the same conditions, was assessed The animal study was reviewed and approved by CEEAH and simultaneously. For this reason, despite the large total number of Generalitat de Catalunya. animals used, the number of each experimental group could not be larger. Whereas the results in females were very consistent, AUTHOR CONTRIBUTIONS this limitation could be considered as compromising behavioral data in those results where intragroup variability would benefit LG-L: conceptualization. TC: performance and analysis of of a bigger sample size. behavior, illustrations, and drafting the manuscript. All authors In conclusion, the present work characterized, for the contributed to the article and approved the submitted version. first time, sex- and age/AD-stage differences in the sensory- discriminative pain threshold to thermal pain stimuli measured FUNDING as the withdrawal response of the left and right hind paws and tail in the plantar test in male and female This work was supported by Instituto de Salud Carlos III, 3xTg-AD mice and as compared to sex- and age-matched ISC3 PI10/00283 to LG-L. It also received support from NTg counterparts with normal aging. We also provided Memorial Mercedes Llort Sender 2021/80/09242021.2. evidence of easy-to-measure emotional dimension with age and sex-depend increased emotional state, suggesting that underlying distinct sex-dependent neurobiological substrate ACKNOWLEDGMENTS derangements are also age-dependent, from young to old age in NTg mice but, most importantly, from premorbid We thank Prof. Frank M. LaFerla Institute for Memory to advanced stages of the disease in the 3xTg-AD mice. Impairments and Neurological Disorders, Department of The results ‘‘blind to sex and age’’ demonstrate the need to Neurobiology and Behavior, University of California, Irvine, analyze both sexes independently. LaFerla’s triple-transgenic USA, for kindly providing the progenitors of the Spanish colonies mice can provide a model for understanding the mechanistic of 3xTg-AD and NTg mice. REFERENCES Berry, A., Capone, F., Giorgio, M., Pelicci, P. G., de Kloet, E. R., Alleva, E., et al. (2007). 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This is an open-access article a risk factor for burns in the elderly. Burns 42, 282–290. doi: 10.1016/j.burns. distributed under the terms of the Creative Commons Attribution License (CC BY). 2015.10.023 The use, distribution or reproduction in other forums is permitted, provided the Jeschke, M. G., and Peck, M. D. (2017). Burn care of the elderly. J. Burn Care Res. original author(s) and the copyright owner(s) are credited and that the original 38, e625–e628. doi: 10.1097/BCR.0000000000000535 publication in this journal is cited, in accordance with accepted academic practice. Kayser, V., Viguier, F., Melfort, M., Bourgoin, S., Hamon, M., and No use, distribution or reproduction is permitted which does not comply with Masson, J. (2015). Altered nociception in mice with genetically induced these terms. Frontiers in Aging Neuroscience | www.frontiersin.org 8 July 2021 | Volume 13 | Article 683412