Intermittent and mild cold stimulation enhances immune function of broilers via co-regulation of CIRP and TRPM8 on NF-κB and MAPK signaling pathways.

Improving immune function is an important indicator for establishing cold adaptation in broilers. In the study, to explore the effects and molecular mechanisms of intermittent and mild cold stimulation (IMCS) on the immune function of broilers, CIRP and TRPM8, induced by cold stimulation, as well as the NF-κB and MAPK pathways which play an important role in immune response, were selected to investigate. A total of 192 one-day-old broilers (Ross 308) were selected and randomly divided into the control group (CC) and the cold stimulation group (CS). The broilers in CC were raised at normal feeding temperature from d 1 to 43, while the broilers in CS were subjected to cold stimulation from day 15 to 35, with a temperature 3 °C below that of the CC group for 5 h, at 1 d intervals. The results showed that IMCS had little effect on the broiler hearts, and the myocardial structure was not damaged. On d 22, IMCS significantly increased the mRNA levels of CIRP, TRPM8, P65, P38, COX-2, TNF-α, IFN- γ, IL-6, IL-10, and the protein levels of CIRP, P65, P38, IL-1ß and iNOS in the hearts, and the levels of CIRP and all cytokines in the serum (P ≤ 0.05). The mRNA and protein levels of IκB-α were significantly reduced (P ≤ 0.05). On d 36, the mRNA levels of TRPM8, P65, ERK, and IL-10 in the hearts and the content of COX-2 in the serum in CS were increased significantly (P ≤ 0.05), while the mRNA levels of IκB-α, P38, and IL-1ß were decreased significantly (P ≤ 0.05). On d 43, IMCS significantly upregulated the mRNA levels of TRPM8, IFN- γ, IL-4, IL-6, IL-10, and the protein levels of IκB-α, P38, and the levels of iNOS, TNF-α, IL6 and IL10 in the serum (P ≤ 0.05); whereas it significantly downregulated CIRP, JNK, P38, iNOS, TNF-α mRNA levels, and CIRP, P65, ERK, JNK, IL1ß and iNOS protein levels (P ≤ 0.05). Therefore, IMCS can enhance broiler immune function through co-regulation of CIRP and TRPM8 on the NF-κB and MAPK pathways, which facilitate the cold adaptation in broilers.

Elevated TRPV2 expression in the facial nerve of rats by cold stimulation: Implications for Bell's palsy.

OBJECTIVE: Bell's palsy, also referred to as clinical manifestations of unilateral facial nerve palsy, encompasses downward angling of the corners of the mouth, the absence of forehead creases, and unilateral incomplete eyelid closure. The incidence of Bell's palsy has increased progressively in recent years, but the underlying mechanism of its occurrence remains unknown; therefore, it is essential to investigate both the cause and treatment of Bell's palsy. Member 2 of the Subfamily V Transient Receptor Potential Cation Channel is a mechanically and thermally sensitive ion channel that plays a crucial role in neural growth and development. Using a novel modeling technique, we endeavored to develop an animal model of Bell's palsy and determine whether TRPV2 expression is altered throughout the course of a facial nerve injury. MATERIALS AND METHODS: The rats were categorized into 3 groups, and their facial nerve function was assessed using RT-qPCR, WB, and pathologic testing, respectively, after undergoing unilateral cold air stimulation for 1, 3, and 7 days. TRPV2 expression was identified using these techniques. RESULTS: In response to cold stimulation, rats exhibited facial nerve paralysis symptoms, demyelinating lesions in the facial nerve, and increased TRPV2 expression. CONCLUSIONS: Extended cold stimulation of the facial nerve in rats may lead to an imbalance in facial nerve homeostasis and increased TRPV2 expression. These findings will contribute to the understanding of the potential mechanism by which cold stimulation affects the facial nerve. Moreover, this finding implies that TRPV2 could possibly function as an additional diagnostic marker or therapeutic target in the context of Bell's palsy.

Intermittent mild cold acclimation ameliorates intestinal inflammation and immune dysfunction in acute cold-stressed broilers by regulating the TLR4/MyD88/NF-κB pathway.

To investigate the potential protective effect of prior cold stimulation on broiler intestine induced by acute cold stress (ACS). A total of 384 one-day-old broilers were divided into control (CON), ACS, cold stimulation Ⅰ (CS3+ACS), and cold stimulation Ⅱ (CS9+ACS) groups. Broilers in CON and ACS groups were reared normally, and birds in CS3+ACS and CS9+ACS groups were reared at 3℃ and 9℃ below CON group for 5 h, respectively, on alternate days from d 15 to 35. Broilers in ACS, CS3+ACS, and CS9+ACS groups were subjected to 10℃ for 24 h on d 43. Eventually, small intestine tissues were collected for histopathological observation and indexes detection. The results showed that intestinal tissues in all ACS-broilers exhibited inflammatory cell infiltrates, microvilli disruption, reduced villus length in jejunum and increased crypt depth in jejunum and ileum. Whereas these phenomena were relatively light in CS3+ACS group. Compared to CON group, mRNA expression of the TLR4/MyD88/NF-κB pathway-related genes (TLR4, MyD88, NF-κBp65, COX-2, iNOS, PTGEs, TNF-α), Th1/Th17-derived cytokines (IL-1ß, IL-2, IL-8, IL-12, IFN-γ, IL-17), and HSPs (HSP40, HSP60, HSP70, HSP90) was upregulated (P < 0.05), and that of Th2-deviated cytokines (IL-4, IL-6, IL-10, IL-13) and IκBα was downregulated (P < 0.05) in small intestine in almost all ACS-broilers. Compared to ACS group, mRNA expression of most of the TLR4/MyD88/NF-κB pathway-related genes, Th1/Th17-derived cytokines, and HSPs was downregulated and that of Th2-derived cytokines was upregulated in CS3+ACS group (P < 0.05). Protein expression levels of TLR4, MyD88, p-p65/p65, p-IκBα/IκBα, IKK, TNF-α, IL-1ß, IL-10, and HSPs were similar to their mRNA expression. The concentration of sIgA and activities of CAT, SOD, and GSH-px were decreased and MDA and H2O2 were increased in ACS and CS9+ACS groups compared to CON group (P < 0.05). Therefore, cold stress caused oxidative stress and inflammation, leading to gut immune dysfunction; while mild cold stimulation at 3℃ below normal rearing temperature alleviated cold stress-induced intestinal injure and dysfunction by modulating the TLR4/MyD88/NF-κB pathway in broilers.

Local alternating heat and cold stimulation affects hemodynamics and oxygenation in fatigued muscle tissue and autonomic nervous activity: a single-arm interventional study.

BACKGROUND: Local alternating heat and cold stimulation as an alternative to contrast bath may cause intermittent vasoconstriction and vasodilation, inducing a vascular pumping effect and consequently promoting increased tissue blood flow and oxygenation. This study aimed to examine the effects of local alternating heat and cold stimulation, using a wearable thermal device, on the hemodynamics of fatigued muscle tissue and autonomic nervous activity. METHODS: Twenty healthy individuals experienced fatigue in the periarticular muscles of the shoulder joint due to a typing task. Local alternating heat and cold stimulations were then applied to the upper trapezius muscle. Muscle hardness was measured using a muscle hardness meter, and muscle tissue hemodynamics and oxygenation were evaluated using near-infrared spectroscopy before and after the stimulation. Autonomic nervous activity was also evaluated using heart rate variability. RESULTS: Alternating heat and cold stimulation decreased muscle hardness of the fatigued trapezius muscle from 1.38 ± 0.15 to 1.31 ± 0.14 N (P < 0.01). The concentration of total hemoglobin in the trapezius muscle tissue increased from - 0.21 ± 1.36 to 2.29 ± 3.42 µmol/l (P < 0.01), and the tissue hemoglobin oxygen saturation also increased from 70.1 ± 5.4 to 71.1 ± 6.0% (P < 0.05). Additionally, the heart rate variability parameter, which is an index of sympathetic nervous activity, increased from 3.82 ± 2.96 to 6.86 ± 3.49 (P < 0.01). A correlation was found between increased tissue hemoglobin oxygen saturation and increased parameters of sympathetic nervous activity (r = 0.50, P < 0.05). CONCLUSIONS: Local alternating heat and cold stimulation affected the hemodynamic response in fatigued muscle tissue and autonomic nervous activity. This stimulation is more efficient than conventional contrast baths in terms of mobility and temperature control and has potential as a new versatile therapeutic intervention for muscle fatigue. TRIAL REGISTRATION: UMIN-CTR (UMIN000040087: registered on April 7, 2020, https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000045710 . UMIN000040620: registered on June 1, 2020, https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000046359 ).

Study on the effect of human sympathetic nerve cold stimulation to relieve driving fatigue based on order recurrence plot.

Driving fatigue is very likely to cause traffic accidents, seriously threatening the lives and properties of drivers. Therefore, accurate detection and effective mitigation of driving fatigue are crucial for ensuring the personal safety of drivers. This study proposes a method to relieve driving fatigue by properly reducing the temperature to stimulate the human sympathetic nerve. The method uses the intelligent cooling and blowing device on the car seat cushion to achieve cold stimulation of the sympathetic nerve of the driver by reducing the temperature of the driver's hip, back and neck, so as to increase the excitement of the sympathetic nerve, keep the driver alert and achieve the purpose of fighting driving fatigue. In view of the fact that the traditional fatigue detection method is easily affected by environmental factors and individual differences, this study uses the order recurrence plot (ORP) method to detect driving fatigue based on electroencephalogram (EEG) signals. The results show that ORP textures drawn by EEG signals of the two driving conditions (normal driving condition and sensory cold stimulation driving condition) are significantly different, and the quantization parameters determinism (DET) and average diagonal line length (DLL) values are significantly different. Cold stimulation of the subjects' hips, back and neck to alleviate driving fatigue was the best when the temperature was 21 °C. In addition, compared with the traditional methods of fatigue relief, the sensory cold stimulation method proposed in this study does not easily to produce tolerance and has no damage to the body.

Effects of intermittent cold stimulation on growth performance, meat quality, antioxidant capacity and liver lipid metabolism in broiler chickens.

Intermittent cold stimulation (ICS) enhances broilers' resistance to cold stress. Nonetheless, further research is needed to investigate the underlying mechanisms that enhance cold stress resistance. A total of 160 one-day-old male Ross 308 broilers were randomly divided into 2 groups (CC and CS5), with the CC group managing temperature according to the standard for broiler growth stages, while the CS5 group were subjected to cold stimulation at a temperature 3℃ lower than the CC group for 5 h, every 2 d from 15 to 35 d. Sampling was conducted at 36 d (36D), 50 d (50D) and after acute cold stress for 24 h (Y24). First, we examined the effects of ICS on broiler growth performance, meat quality, antioxidant capacity, and lipid metabolism. The results demonstrated that ICS enhanced the performance of broilers to a certain degree. Specifically, the average weight gain in the CS5 group was significantly higher than that of the CC group, and the feed conversion ratio significantly decreased compared to CC at 4 W and 6 W (P ≤ 0.05). Compared with the CC group, cold stimulation significantly reduced drip loss, shearing force, and yellowness (a* value) of chicken meat, while significantly increased redness (b* value) (P ≤ 0.05). At Y24, the levels of T-AOC and GSH-PX in the serum of the CS5 group were significantly higher than those of the CC group, while the level of MDA was significantly lower (P ≤ 0.05). The content of TG, FFA, and VLDL in the serum of the CS5 group was significantly elevated, whereas the level of TC and HDL was significantly lower (P ≤ 0.05). In addition, we further explored whether AMPK-mTOR pathway is involved in the regulation of changes in lipid metabolism and the possible regulatory mechanisms downstream of the signaling pathway. The results showed that ICS significantly upregulated the expression levels of AMPK mRNA and protein in the liver of the CS5 group at 36D and Y24, while significantly down-regulating mTOR (P ≤ 0.05). Compared with the CC group, ICS significantly down-regulated the mRNA expression levels of lipid synthesis and endoplasmic reticulum stress-related genes (SREBP1c, FAS, SCD, ACC, GRP78 and PERK) at 36D and Y24, while significantly up-regulating the mRNA expression levels of lipid decomposition and autophagy-related genes (PPAR and LC3) (P ≤ 0.05). In addition, at Y24, the protein expression levels of endoplasmic reticulum stress-related genes (GRP78) in the CS5 group were significantly lower, while autophagy-related genes (LC3 and ATG7) were significantly higher (P ≤ 0.05). ICS can affect meat quality and lipid metabolism in broilers, and when broilers are subjected to acute cold stress, broilers trained with cold stimulation have stronger lipid metabolism capacity.

Preliminary study of finger temperature recovery in patients with diabetes mellitus following cold stimulation.

OBJECTIVE: To explore the difference in temperature recovery following cold stimulation between participants with and without diabetes mellitus (DM). MATERIALS AND METHODS: The participants without (control group; n = 25) and with (DM group; n = 26) DM were subjected to local cold stimulation (10º C for 90 s). The thermal images of their hands were continuously captured using a thermal camera within 7 min following cold stimulation, and the highest temperature of each fingertip was calculated. According to the temperature values at different timepoints, the temperature recovery curves were drawn, and the baseline temperature (T-base), initial temperature after cooling (T0), temperature decline amplitude (T-range), and area under the temperature recovery curve > T0 (S) were calculated. Finally, symmetry differences between the two groups were analysed. RESULTS: No statistical differences in the T-base, T0, and T-range were observed between the DM and control groups. After drawing the rewarming curve according to the temperature of the fingertips of the patients following cold stimulation, the S in the DM group was significantly lower than that in the control group (p < 0.05). Furthermore, the asymmetry of the base temperature of the hand was observed in the DM group. CONCLUSIONS: Following cold stimulation, the patients with DM exhibited a different rewarming pattern than those without DM. Thus, cold stimulation tests under infrared thermography may contribute to the early screening of diabetic peripheral neuropathy in future.

Intermittent mild cold stimulation alleviates cold stress-induced pulmonary fibrosis by inhibiting the TGF-ß1/Smad signaling pathway in broilers.

To investigate the potential protective effect of intermittent cold stimulation on lung tissues of broilers exposed to acute cold stress (ACS). A total of 384 one-day-old broilers were assigned to 4 experimental groups with 6 replicates of 16 birds each: control (CON) and ACS groups were reared at normal feeding temperature from d 1 to 42; cold treatment groups (CS3+ACS and CS9+ACS) were reared, respectively, at 3°C or 9°C for 5 h on alternate days below the CON group from d 15 to 35. Animals in CS3+ACS, CS9+ACS, and ACS groups were exposed at 10°C for 24 h on d 43. Subsequently, lung tissues were collected to perform histopathological examination and measurement of relevant indexes. The results showed that lung tissues in CS9+ACS and ACS groups exhibited increased inflammatory cell infiltrates and collagen deposition compared to the CON group, while this pathological phenomenon was less pronounced in the CS3+ACS group. Compared to CON group, H2O2 and MDA contents were increased, and the activities of antioxidant enzymes (CAT, SOD, GPx, T-AOC) were reduced in CS9+ACS and ACS group (P < 0.05); mRNA and protein levels of inhibitor of NF-κB, Smad7, matrix metallopeptidase (MMP)-2, MMP9, and antioxidant-related genes were downregulated, whereas mRNA and protein levels of genes related to NF-κB/NLRP3 pathway-regulated inflammation and TGF-ß1/Smad pathway-regulated fibrosis were upregulated in cold-stressed broilers (P < 0.05). mRNA levels of heme oxygenase-1, NAD(P)H quinone oxidoreductase-1, and MMP9 were increased in CS3+ACS group (P < 0.05). Moreover, the expression of most antioxidant-related genes was increased, and that of inflammation- and fibrosis-related genes was reduced in CS3+ACS group (P < 0.05). Therefore, cold stress caused oxidative stress and inflammation, leading to pulmonary fibrosis in broilers, whereas intermittent mild cold stimulation at 3°C below normal rearing temperature alleviated fibrosis by inhibiting the TGF-ß1/Smad pathway modulated by the Nrf2/HO-1 and NF-κB/NLRP3 signaling pathway. This study suggests that intermittent mild cold stimulation can be a potential strategy to reduce ACS-induced lung damage in broilers.

Intermittent cold stimulation affects energy metabolism and improves stress resistance in broiler heart.

To investigate the effect of intermittent cold stimulation on cardiac energy metabolism and cold resistance of broilers, 288 broilers were divided into 3 groups: control group (CC) and 2 cold stimulation groups (CS3 and CS9). The CS3 and CS9 groups received cold stimulation at temperatures of 3°C and 9°C lower than CC group for 5 h from d 15 to 35. Three groups were subjected to acute cold stress (ACS) of 10°C for 12 and 24 h at 44 d. Performance, cardiac histopathological changes, heat shock proteins (HSPs), and lipid metabolism levels were measured. Results showed that the performance was not different among groups at 22 and 29 d (P > 0.05), but the mRNA levels of Acyl CoA synthase long-chain family member 1 (ACSL1) and acyl-coenzyme oxidase (ACO) in CS group were upregulated compared to CC group (P < 0.05). At 36 d, the performance of the CS3 group was better than the other 2 groups, myocardial structure was normal and other lipid metabolism indexes, except for peroxisome proliferator-activated receptor coactivator 1α (PGC-1α) levels, were similar to those of CC group (P > 0.05). The myocardial fiber disorder, Triglyceride (TG), and leptin (LEP) contents were significantly lower in CS9 group than in CC and CS3 groups at 36 d (P < 0.05). The HSP protein levels were significantly higher in CS group than in CC group before ACS (P < 0.05). After 24 h of ACS, the mRNA of lipid metabolism genes, the protein levels of HSP40 and HSP60, and the contents of TG and LEP in the CS3 group were upregulated compared to other groups. The CC and CS9 groups showed myocardial structure was destroyed, with lower TG and LEP levels compared to before ACS (P < 0.05). Therefore, cold stimulation at 3°C lower than the normal feeding temperature for 5 h did not impair performance but can increase the resistance of broilers to ACS by promoting lipid metabolism.

Cold stimulation causes oxidative stress, inflammatory response and apoptosis in broiler heart via regulating Nrf2/HO-1 and NF-κB pathway.

To investigate the effect of cold stimulation on heart, 300 1-day-old female broilers were divided into control (CON) and two cold stimulation (CS3 and CS9) groups. Birds in CON group were reared in normal ambient temperature during day 1-43; while birds in CS3 and CS9 groups were reared at 3 °C and 9 °C below CON group for 5 h at 1-day intervals from day 15 to day 35, respectively. Heart tissues were collected at day 22, 29, 36, and 43 to determine the indexes related to oxidative stress, inflammation and apoptosis. The H&E staining displayed that inflammatory cell infiltration and myocardial fiber break were obviously observed in CS9 group, and cardiac pathological score in CS9 group was higher than CON and CS3 groups (P < 0.05) at day 22, 36, and 43. Overall, compared to CON group, the concentrations of MDA and H2O2 were elevated, the activities of SOD, CAT, GPx, and T-AOC were reduced, and mRNA expression of CAT, GPx, SOD, nuclear factor-erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) was downregulated in CS9 group at each time-point (P < 0.05). Compared to CON group, mRNA expression of NF-κBp65, COX-2, iNOS, PTGEs, TNF-α, and IL-1ß, and mRNA and protein expression of Bax, Bak, Cyt-c, caspase-3, and caspase-9 were increased, while Bcl-2 and Bcl-2/Bax ratio were decreased in CS9 group (P < 0.05) at the most detected time-points. There were no significant differences in the levels of indexes associated with oxidative stress, Nrf2/HO-1 antioxidant system, inflammation, and apoptosis between CON and CS3 groups at the most detected time-points (P > 0.05). Therefore, this study suggests that severe cold stimulation at 9 °C below normal rearing temperature induces cardiomyocyte inflammation and apoptosis by regulating Nrf2/HO-1 pathway-related oxidative stress in broilers, and mild cold stimulation of CS3 group can improve the adaptability of hearts to cold environment.

Appropriate cold stimulation changes energy distribution to improve stress resistance in broilers.

Appropriate cold stimulation can improve stress resistance in broilers and alleviate the adverse impacts of a cold environment. To investigate the effects of intermittent mild cold stimulation (IMCS) on energy distribution in the livers of broilers, 96 healthy 1-d-old Ross-308 male broilers were randomly divided into the control group (CC) and the cold stimulation group (H5). The CC group was raised at a normal thermal temperature, i.e., 35 °C until 3 d, after which the temperature was dropped gradually by 0.5 °C/d until 20 °C at 33 d. This temperature was maintained until 49 d. The H5 group was raised at the same temperature as the CC group until 14 d (35 to 29.5 °C) and at 3 °C below the temperature of the CC group starting at 0930 hours for 5 h every other day from 15 to 35 d (26 to 17°C). The temperature was returned to 20 °C at 36 d and maintained until 49 d. At 50 d, all broilers were subjected to acute cold stress (ACS) at 10 °C for 6 and 12 h. We found that IMCS had positive effects on production performance. Using transcriptome sequencing of the broiler livers, 327 differentially expressed genes (DEG) were identified, and highly enriched in fatty acid biosynthesis, fatty acid degradation, and the pyruvate metabolism pathway. When compared to the CC group, the mRNA levels of ACAA1, ACAT2, ACSL1, CPT1A, LDHB, and PCK1 in the H5 group were increased at 22 d (P < 0.05). The LDHB mRNA level was upregulated in the H5 group at 29 d compared to the CC group (P < 0.05). After 21 d of IMCS (at 36 d), the mRNA expression levels of ACAT2 and PCK1 were found to be significantly increased in the H5 group compared to the CC group (P < 0.05). Seven days after the IMCS had ended (at 43 d), the mRNA levels of ACAA1, ACAT2, and LDHB in the H5 group were higher than in the CC group (P < 0.05). The mRNA levels of heat shock protein (HSP) 70, HSP90, and HSP110 in the H5 group were higher than in the CC group after 6 h of ACS (P < 0.05). The protein levels of HSP70 and HSP90 in the H5 group were downregulated after 12 h of ACS, compared to the CC group (P < 0.05). These results indicated that IMCS at 3 °C lower than the normal temperature could improve energy metabolism and stress resistance in the livers of broilers, alleviate the damage of short-term ACS on broilers, help broilers adapt to the low temperature, and maintain stable of energy metabolism in the body.

Cold stimulation has a great impact on broilers. Excessive cold stimulation can lead to damage, while intermittent mild cold stimulation (IMCS) can make broilers adapt to the cold environment. Low temperature will make the body produce lots of heat to maintain metabolic stability. The liver controls the energy metabolism, and the avian livers are the main organ regulating lipid metabolism. In this study, the broilers of different ages were subjected to cold stimulation training and then acute cold stress (ACS). We found that IMCS had positive effects on production performance. Through transcriptome sequencing, we found that the differentially expressed genes were highly enriched in the energy metabolism pathway, and the expression levels of the most key genes and heat shock proteins were upregulated. The stress resistance was also enhanced, which could alleviate the damage of short-term ACS to the body. The broilers gradually adapted to the low-temperature environment and finally established cold adaptation. The findings of this work will be helpful to the development of animal husbandry in cold regions and improve animal welfare.

Thermogenic Capacity of Human Supraclavicular Brown Fat and Cold-Stimulated Brain Glucose Metabolism.

Human brain metabolism is susceptible to temperature changes. It has been suggested that the supraclavicular brown adipose tissue (BAT) protects the brain from these fluctuations by regulating heat production through the presence of uncoupling protein 1 (UCP-1). It remains unsolved whether inter-individual variation in the expression of UCP-1, which represents the thermogenic capacity of the supraclavicular BAT, is linked with brain metabolism during cold stress. Ten healthy human participants underwent 18F-FDG PET scanning of the brain under cold stimulus to determine brain glucose uptake (BGU). On a separate day, an excision biopsy of the supraclavicular fat-the fat proximal to the carotid arteries supplying the brain with warm blood-was performed to determine the mRNA expression of the thermogenic protein UCP-1. Expression of UCP-1 in supraclavicular BAT was directly related to the whole brain glucose uptake rate determined under cold stimulation (rho = 0.71, p = 0.03). In sub-compartmental brain analysis, UCP-1 expression in supraclavicular BAT was directly related to cold-stimulated glucose uptake rates in the hypothalamus, medulla, midbrain, limbic system, frontal lobe, occipital lobe, and parietal lobe (all rho ≥ 0.67, p < 0.05). These relationships were independent of body mass index and age. When analysing gene expressions of BAT secretome, we found a positive correlation between cold-stimulated BGU and DIO2. These findings provide evidence of functional links between brain metabolism under cold stimulation and UCP-1 and DIO2 expressions in BAT in humans. More research is needed to evaluate the importance of these findings in clinical outcomes, for instance, in examining the supporting role of BAT in cognitive functions under cold stress.

Effect of intermittent mild cold stimulation on intestinal immune function and the anti-stress ability of broilers.

A total of 240 healthy 1-day-old Ross 308 male broilers were randomly divided into 3 groups (CS0 group, CS3 group, and CS6 group), with 5 replicates in each group and 16 broilers in each replicate, in order to evaluate the effects of intermittent mild cold stimulation (IMCS) on the intestinal immune function and anti-cold stress ability of broilers after acute cold stress. The mRNA expression levels of cytokines and Toll-like receptors (TLRs) in the duodenum and jejunum were detected at the end of cold stimulation (36 d), 2 wk after recovery (50 d), and after acute cold stress (Y6). In addition, the mRNA and protein expression levels of heat shock proteins (HSPs) were measured before and after acute cold stress. The experimental data were statistically processed using 1-way ANOVA and Duncan's multiple comparisons. The results showed that the mRNA expression levels of IL2, IL8, IFN γ, TLR7, and TLR21 in the duodenum and IL2 and IFN Î³ in jejunum were significantly higher in the CS6 group than in the CS0 and CS3 groups at 36 d (P < 0.05). All TLR levels in the jejunum were significantly lower in the CS3 group than in the CS0 and CS6 groups at 36 d (P < 0.05). After 6 h of acute cold stress, in the duodenum, the mRNA expression levels of IL6 and IL8 were significantly decreased in the CS0 and CS6 groups compared to levels at 50 d (P < 0.05), while levels in the CS3 group remained stable (P > 0.05). Compared with 50 d, the expression level of HSP mRNA in the jejunum in the CS3 group was relatively stable compared to that in the CS0 and CS6 groups after acute cold stress (P > 0.05). At the protein level, the HSP60 expression level in the duodenum and HSP40, HSP60, and HSP70 expression levels in the jejunum were significantly higher in the CS3 group than in the CS0 and CS6 groups after acute cold stress (P < 0.05). In conclusion, cold stimulation training at 3℃/3 h lower than the conventional feeding temperature can improve the intestinal immune function and anti-stress ability of broilers.

Effect of cold stimulation on phenotype of alveolar macrophages in mice / 中国生物制品学杂志

@#Objective To investigate the effect of cold stimulation on the phenotype of alveolar macrophages(MH-S cells) in mice. Methods MH-S cells were cultured at 37 ℃ for 24 h,and cold stimulated at 36,34 and 32 ℃ for 0,0. 5,1,3,6,9 and 12 h respectively. The mRNA transcription levels of interleukin-1β(IL-1β) and interleukin-10(IL-10) genes in MH-S cells were detected by qRT-PCR. MH-S cells were cultured at 37 ℃ for 24 h,and cold stimulated at 34 ℃ for 0. 5 h,which were detected for the mRNA transcription levels of tumor necrosis factor-α(TNF-α),inducible nitric oxide synthase(iNOS)and Arginase1(Arg1)genes by qRT-PCR(MH-S cells with 0 h cold stimulation as control),detected for the expression of iNOS and Arg1 by immunofluorescence assay(MH-S cells cultured at 37 ℃ for 0. 5 h as negative control)and detected for the expression levels of iNOS,TNF-α and nuclear factor-kappa B(NF-κB)by Western blot(MH-S cells cultured at 37 ℃ for 0. 5 h as negative control). Results The mRNA transcription levels of IL-1β and IL-10 genes in MH-S cells were the highest when the cells were cultured at 34 ℃ for 0. 5 h,therefore,the cold stimulation model of MH-S cells was established under this condition. Compared with the cells cultured for 0 h,the mRNA transcription levels of iNOS,TNF-α and Arg1genes in MH-S cells cultured at 34 ℃ for 0. 5 h increased significantly(t = 3. 733,12. 190 and 6. 793,respectively,each P < 0. 05). Compared with the negative control group,the fluorescence expression intensity of iNOS and Arg1 in MH-S cells in the stimulation group increased,especially iNOS,the expression levels of iNOS and TNF-α proteins increased with no significant difference(t = 0. 675 and 1. 514,respectively,each P > 0. 05),and the expression level of NF-κB increased significantly(t = 3. 092,P < 0. 05). Conclusion Cold stimulation at 34 ℃ for 0. 5 h can increase the expression of inflammatory factors such as IL-1β,IL-10,TNF-α,iNOS,Agr1 and NF-κB in MH-S cells,activate NF-κB signaling pathway in MH-S cells,induce the expression of inflammatory proteins and promote cell activation.

Effects of Acute Cold Stress after Intermittent Cold Stimulation on Immune-Related Molecules, Intestinal Barrier Genes, and Heat Shock Proteins in Broiler Ileum.

Cold stress will have a negative impact on animal welfare and health. In order to explore the effect of intermittent cold stimulation training on the cold resistance of broilers. Immune-related and intestinal barrier genes were detected before and after acute cold stress (ACS), aiming to find an optimal cold stimulation training method. A total of 240 1-day-old Ross broilers (Gallus) were divided into three groups (G1, G2, and G3), each with 5 replicates (16 chickens each replicate). The broilers of G1 were raised at normal temperature, while the broilers of G2 and G3 were treated with cold stimulation at 3 °C lower than the G1 for 3 h and 6 h from 15 to 35 d, respectively, at one-day intervals. At 50 d, the ambient temperature for all groups was reduced to 10 °C for six hours. The results demonstrated that before ACS, IL6, IL17, TLR21, and HSP40 mRNA levels in G3 were apparently down-regulated (p < 0.05), while IL8 and Claudin-1 mRNA levels were significantly up-regulated compared with G1 (p < 0.05). After ACS, IL2, IL6, and IL8 expression levels in G3 were lower than those in G2 (p < 0.05). Compared to G2, Claudin-1, HSP90 mRNA levels, HSP40, and HSP70 protein levels were increased in G3 (p < 0.05). The mRNA levels of TLR5, Mucin2, and Claudin-1 in G2 and IL6, IL8, and TLR4 in G3 were down-regulated after ACS, while IL2, IL6, and IL17 mRNA levels in G2 and HSP40 protein levels in G3 were up-regulated after ACS (p < 0.05). Comprehensive investigation shows that cold stimulation at 3 °C lower than the normal feeding temperature for six hours at one day intervals can enhanced immune function and maintain the stability of intestinal barrier function to lessen the adverse effects on ACS in broilers.

Effect of mild intermittent cold stimulation on thymus immune function in broilers.

This study aims to assess the effect of intermittent and mild cold stimulation (IMCS) on thymus function and the ability of 1-day-old male Ross 308 broilers to withstand cold. Four hundred broilers were reared under normal and mild cold temperatures at 3°C below the normal feeding temperature and were subjected to acute cold stress (ACS) at 10°C on d 50 at 7 am for 6 h, 12 h, and 24 h. We determined the expression levels of toll-like receptors (TLRs), cytokines and avian ß-defencins (AvBDs), encoding genes in thymus of broilers at 22, 36, 43, and 50 d of age, and the serum ACTH and cortisol (CORT) levels at 50 d of age. At D22 and D36, the mRNA expression levels of TLRs and AvBDs genes in CS groups were generally significantly decreased (P < 0.05). The lowest expression levels were found in birds submitted to intermittent and mild cold stimulation training for 5 h (CS5 group) on d 22 and 36 of development (P < 0.05). At D43 and D49 after IMCS, mRNA expression levels of most TLRs and AvBDs were significantly lower than those in CC group (P < 0.05), and that mRNA expression levels of all TLRs and most AvBDs in CS5 group had the same change trend with age as those in CC group (P > 0.05). At D22 and D36, mRNA expression levels of different cytokines in each CS groups were different (P < 0.05). mRNA expression levels of IL-2, IL-4, IL-6, IL-8, IL-17, and IFN-α all reached the highest values in the CS5 group at D36 (P < 0.05). The levels of ACTH and CORT in all IMCS-treated birds changed in varying degrees after ACS, but there was no significant change in CS5 group (P > 0.05). Collectively, different cold stimulation schemes could modulate thymus immune function of broilers by maintaining homeostasis and enhancing cold resistance. In particular, the optimal cold adaptation scheme was at 3°C below the conventional feeding temperature for 5 h.

Adipose Tissue Myeloid-Lineage Neuroimmune Cells Express Genes Important for Neural Plasticity and Regulate Adipose Innervation.

Peripheral nerves allow a bidirectional communication between brain and adipose tissues, and many studies have clearly demonstrated that a loss of the adipose nerve supply results in tissue dysfunction and metabolic dysregulation. Neuroimmune cells closely associate with nerves in many tissues, including subcutaneous white adipose tissue (scWAT). However, in scWAT, their functions beyond degrading norepinephrine in an obese state remain largely unexplored. We previously reported that a myeloid-lineage knockout (KO) of brain-derived neurotrophic factor (BDNF) resulted in decreased innervation of scWAT, accompanied by an inability to brown scWAT after cold stimulation, and increased adiposity after a high-fat diet. These data underscored that adipose tissue neuroimmune cells support the peripheral nerve supply to adipose and impact the tissue's metabolic functions. We also reported that a subset of myeloid-lineage monocyte/macrophages (Ly6c+CCR2+Cx3cr1+) is recruited to scWAT in response to cold, a process known to increase neurite density in adipose and promote metabolically healthy processes. These cold-induced neuroimmune cells (CINCs) also expressed BDNF. Here we performed RNAseq on CINCs from cold-exposed and room temperature-housed mice, which revealed a striking and coordinated differential expression of numerous genes involved in neuronal function, including neurotrophin signaling and axonal guidance, further supporting that CINCs fulfill a nerve-supporting role in adipose. The increased expression of leukocyte transendothelial migration genes in cold-stimulated CINCs also confirms prior evidence that they are recruited to scWAT and are not tissue resident. We now provide whole-depot imaging of scWAT from LysM-BDNF KO mice, revealing a striking reduction of innervation across the depot fitting with their reduced energy expenditure phenotype. By contrast, Cx3cr1-BDNF KO mice (a macrophage subset of LysM+ cells) exhibited increased thermogenesis and energy expenditure, with compensatory increased food intake and no change in adiposity or body weight. While these KO mice also exhibit a significantly reduced innervation of scWAT, especially around the subiliac lymph node, they displayed an increase in small fiber sympathetic neurite branching, which may underlie their increased thermogenesis. We propose a homeostatic role of scWAT myeloid-lineage neuroimmune cells together in nerve maintenance and neuro-adipose regulation of energy expenditure.

Effects of alternating heat and cold stimulation at different cooling rates using a wearable thermo device on shoulder muscle stiffness: a cross-over study.

BACKGROUND: A small, wearable thermo device that uses Peltier elements for programmed heat and cold stimulation has been developed recently and is expected to be applied in conventional contrast bath therapy. This study was aimed to examine improvements in trapezius muscle hardness and subjective symptoms resulting from alternating heat and cold stimulation, with different rates of cooling. METHODS: This cross-over study included four conditions. Twenty healthy young male individuals (age, 22.3 ± 4.5 years) participated in this study. These four interventions targeted the unilateral trapezius muscle of the dominant arm after a 15-min typing task. Specifically, heat and cold stimulations were applied at different ratios (the heating/cooling rate of 3:1, 3:2, and 3:3) or not applied. Each intervention was separated by at least one week. Skin temperature at the stimulation area was recorded using a data logger. Outcome measures included muscle hardness (measured using a portable tester) and subjective symptoms (muscle stiffness and fatigue). Each item was assessed at three time points: baseline, after typing, and after the intervention. RESULTS: Two-way analysis of variance with repeated measures found an interaction effect for muscle hardness between four conditions (3:1, 3:2, 3:3, and no) and three time points (p < 0.05). Only in the 3:1 condition were the post-intervention values lower than those after typing (p < 0.01). There was also an interaction effect for subjective muscle stiffness (p < 0.05); the values after the intervention in the 3:1 condition were lower than those after intervention in the no stimulation condition (p < 0.01). There was no significant relationship between changes in muscle hardness and changes in subjective symptoms in the 3:1 condition. CONCLUSIONS: Our results demonstrate that alternating heat and cold stimulations with a different cooling rate could affect the degree of improvement in muscle hardness and subjective symptoms. In particular, the 3:1 condition has the possibility to improved muscle hardness within the condition and subjective muscle stiffness between conditions. TRIAL REGISTRATION: UMIN000040620. Registered 1 June 2020, https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000046359.

Electroencephalographic Measurement on Post-stroke Sensory Deficiency in Response to Non-painful Cold Stimulation.

Background: Reduced elementary somatosensation is common after stroke. However, the measurement of elementary sensation is frequently overlooked in traditional clinical assessments, and has not been evaluated objectively at the cortical level. This study designed a new configuration for the measurement of post-stroke elementary thermal sensation by non-painful cold stimulation (NPCS). The post-stroke cortical responses were then investigated during elementary NPCS on sensory deficiency via electroencephalography (EEG) when compared with unimpaired persons. Method: Twelve individuals with chronic stroke and fifteen unimpaired controls were recruited. A 64-channel EEG system was used to investigate the post-stroke cortical responses objectively during the NPCS. A subjective questionnaire of cold sensory intensity was also administered via a numeric visual analog scale (VAS). Three water samples with different temperatures (i.e., 25, 10, and 0°C) were applied to the skin surface of the ventral forearm for 3 s via glass beaker, with a randomized sequence on either the left or right forearm of a participant. EEG relative spectral power (RSP) and topography were used to evaluate the neural responses toward NPCS with respect to the independent factors of stimulation side and temperature. Results: For unimpaired controls, NPCS initiated significant RSP variations, mainly located in the theta band with the highest discriminative resolution on the different temperatures (P < 0.001). For stroke participants, the distribution of significant RSP spread across all EEG frequency bands and the temperature discrimination was lower than that observed in unimpaired participants (P < 0.05). EEG topography showed that the NPCS could activate extensive and bilateral sensory cortical areas after stroke. Significant group differences on RSP intensities were obtained in each EEG band (P < 0.05). Meanwhile, significant asymmetry cortical responses in RSP toward different upper limbs were observed during the NPCS in both unimpaired controls and participants with stroke (P < 0.05). No difference was found between the groups in the VAS ratings of the different temperatures (P > 0.05). Conclusion: The post-stroke cortical responses during NPCS on sensory deficiency were characterized by the wide distribution of representative RSP bands, lowered resolution toward different temperatures, and extensive activated sensory cortical areas.

Effects of alternating heat and cold stimulation using a wearable thermo-device on subjective and objective shoulder stiffness.

BACKGROUND: Technological innovations have allowed the use of miniature apparatus that can easily control and program heat and cold stimulations using Peltier elements. The wearable thermo-device has a potential to be applied to conventional contrast bath therapy. This study aimed to examine the effects of alternating heat and cold stimulation (HC) using a wearable thermo-device on subjective and objective improvement of shoulder stiffness. METHODS: Twenty healthy young male individuals (20.3 ± 0.6 years) participated in this study. The interventions were randomly conducted under four conditions, including HC, heat stimulation, cold stimulation, and no stimulation on their bilateral trapezius muscle, after a 30-min typing task. Each intervention was administered at least 1 week apart. The analyzed limb was the dominant arm. Muscle hardness was assessed using a portable muscle hardness meter, as well as the skin temperature over the stimulated area. After each condition, the participants were asked for feedback regarding subjective improvement in refreshed feelings, muscle stiffness, and muscle fatigue using an 11-point numerical rating scale. RESULTS: With regard to muscle hardness, only the HC condition significantly decreased from 1.43 N to 1.37 N (d = 0.44, p < 0.05). Additionally, reduced muscle hardness in HC condition was associated with the degree of skin cooling during the intervention (cold max: r = 0.634, p < 0.01; cold change: r = -0.548, p < 0.05). Subjective improvement in refreshed feelings, muscle stiffness, and muscle fatigue was determined in the HC and heat stimulation conditions compared with the no stimulation condition (p < 0.01 and p < 0.05, respectively). Moreover, the HC condition showed significantly greater improvements in muscle stiffness and fatigue compared to the cold stimulation condition (p < 0.05). CONCLUSIONS: The current study demonstrated that HC promoted not only better subjective symptoms, such as muscle stiffness and fatigue, but also lesser muscle hardness. Furthermore, an association was observed between the degree of skin temperature cooling and reduced muscle hardness during HC. Further investigations on the ratio and intensity of cooling should be conducted in the future to establish the optimal HC protocol for muscle stiffness or fatigue. TRIAL REGISTRATION: UMIN000040620 . Registered 1 June 2020.