Serum and plasma brain-derived neurotrophic factor concentration are elevated by systemic but not local passive heating.

Brain-derived neurotrophic factor (BDNF) plays a key role in neuronal adaptations. While previous studies suggest that whole-body heating can elevate circulating BDNF concentration, this is not known for local heating protocols. This study investigated the acute effects of whole-body versus local passive heating on serum and plasma BDNF concentration. Using a water-perfused suit, ten recreationally active males underwent three 90 min experimental protocols: heating of the legs with upper-body cooling (LBH), whole-body heating (WBH) and a control condition (CON). Blood samples were collected before, immediately after and 1 h post-heating for the determination of serum and plasma BDNF concentration, platelet count as well as the BDNF release per platelet. Rectal temperature, cardiac output and femoral artery shear rate were assessed at regular intervals. Serum and plasma BDNF concentration were elevated after WBH (serum: 19.1±5.0 to 25.9±11.3 ng/ml, plasma: 2.74±0.9 to 4.58±2.0; p<0.044), but not LBH (serum: 19.1±4.7 to 22.3±4.8 ng/ml, plasma: 3.25±1.13 to 3.39±0.90 ng/ml; p>0.126), when compared with CON (serum: 18.6±6.4 to 16.8±3.4 ng/ml, plasma: 2.49±0.69 to 2.82±0.89 ng/ml); accompanied by an increase in platelet count (p<0.001). However, there was no change in BDNF content per platelet after either condition (p = 0.392). All physiological measures were elevated to a larger extent after WBH compared with LBH (p<0.001), while shear rate and rectal temperature were higher during LBH than CON (p<0.038). In conclusion, WBH but not LBH acutely elevates circulating BDNF concentration. While these findings further support the use of passive heating to elevate BDNF concentration, a larger increase in shear rate, sympathetic activity and/or rectal temperature than found after LBH appears needed to induce an acute BDNF response by passive heating.

Introduction

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family that plays a key role in neuroplasticity, learning and memory as well as metabolic regulation [1]. BDNF knock-out mice show impaired spatial learning, reduced survival of neurons in the hippocampus [2] and develop obesity [3]. Furthermore, blocking the BDNF receptor tropomyosin receptor kinase B (TrkB) attenuates improvements in cognitive function following exercise training in rats [4]. The strong correlation of BDNF expressed in brain structures such as the hippocampus with circulating BDNF concentration provides the opportunity to investigate BDNF expression in humans [5]. As such, an increasing number of studies have investigated strategies to elevate circulating BDNF concentration as well as their potential to treat or prevent conditions related to neuronal impairments such as Alzheimer`s disease, depression and schizophrenia [6].

Acute and chronic aerobic exercise can elevate circulating BDNF concentration in humans [7]. However, physical exercise is not universally accessible to all members of society, due to for instance disability, chronic disease or cognitive impairments. Passive heat treatment is a promising intervention strategy that may benefit systemic health, especially in situations where exercise is not accessible [8, 9]. In the context of elevating BDNF concentration, a single bout of aerobic exercise in the heat induces a larger acute BDNF response compared to exercise in a thermoneutral environment [10]. Moreover, 20 min of whole-body immersion in 42°C water also acutely elevates serum BDNF concentration [11]. In support of this acute effect, a recent trial found that 10 weeks of repeated head-out, dry hyperthermic exposure significantly increased serum BDNF concentration in young adults [12]. However, as with exercise, the cardiovascular and heat strain induced by whole-body passive heating may preclude some individuals from engaging in this activity [13]. For instance, people with chronic heart failure may be advised against engagement in whole-body heating due to the attendant cardiovascular strain [14], while the impaired thermoregulation in older adults and persons with type II diabetes mellitus may place them at an increased risk for heat-illness during intense heat stress [15, 16]. As such, prior to promoting passive heating as a strategy to stimulate BDNF-mediated improvements in cognitive function, more accessible and physiologically less strenuous protocols may provide an additional tool to promote health in persons for whom whole-body heating may be contraindicated.

The primary cellular sources of circulating BDNF are suggested to be the brain, vascular endothelial cells, and peripheral blood mononuclear cells. A small proportion of circulating BDNF is unbound and freely interacts with TrkB, whereas the majority of circulating BDNF is bound to platelets [17]. Increased BDNF in response to hyperthermia may be mediated through multiple mechanisms, including increased in shear stress, stimulating BDNF release by endothelial cells and platelets [18, 19]; an increase in the permeability of the blood-brain-barrier with an increase in body temperature, increasing the contribution of BDNF from the brain [11]; or a sympathetic activation-mediated increase in the release of BDNF containing platelets from the spleen (thrombocytosis) [20]. These suggested drivers of the acute BDNF response following heat stress indicate that systemic hyperthermia may not be essential to elevate BDNF concentration, as for instance blood flow can also be increased through localised heating [21]. Apart from a reduced cardiovascular strain, local passive heating has recently also been shown to result in more favourable perceptual responses compared with whole-body passive heating [22]; potentially positively affecting uptake and adherence to passive heating interventions [23]. However, the effect of local heating on the circulating BDNF concentration has yet to be determined.

Therefore, this study compared the effects of acute whole-body heating (WBH) versus lower-limb heating in combination with cooling of the upper body (LBH) on circulating BDNF and cardiovascular strain in young, healthy adults. It was hypothesised that the LBH would evoke lower cardiovascular strain compared with WBH, but would nonetheless elevate circulating BDNF concentration to a similar magnitude as WBH due to increases in shear stress and sympathetic nerve activity.

Materials and methods

Ten healthy young males (age: 24±3 yrs; height: 184±6 cm; body mass: 80±15 kg; BMI: 23.0±4.9 kg/m2; body fat percentage: 15.7±4.2%) participated in this study after providing written informed consent. Exclusion criteria were smoking and the use of anti-inflammatory medication. This study reports secondary findings from a larger trial that investigated the effect of LBH versus WBH on inflammation, glycaemic and perceptual responses [21]. As such, the participants and heating protocols described herein are identical to Hoekstra et al. [21]. The study procedures were approved by the ethics committee of Loughborough University (project code: R19-P084), according to the declaration of Helsinki.

Study design

Participants visited the laboratory following an overnight fast on three occasions, separated by at least 72 h. Participants avoided exercise and the consumption of caffeine and alcohol on the day prior to their laboratory visits. In addition, they monitored their food and drink consumption prior to the first laboratory visit and adhered to the same diet on the day before the following visits. The heating protocols used have been described in detail previously [21]. Briefly, body temperature was manipulated using a water-perfused suit (Med-Eng, Ottawa, Canada), with separate controls for the lower and upper body segments. Participants undertook three 90 min experimental conditions in a randomised order: 1) whole-body heating (WBH), where 50°C water was perfused through the upper and lower body part of the suit; 2) lower-body heating with simultaneous cooling of the upper body (LBH), where 50°C water was perfused through the lower body part of the suit and upper-body cooling was applied by cool packs and 2°C water perfused through the upper body part of the suit; 3) a control condition (CON), where 36°C water was perfused through both parts of the suit. Ambient temperature and relative humidity in the laboratory were 24.4±0.6°C and 44±8% during CON, 24.6±0.7°C and 43±9% during LBH, and 24.5±1.0°C and 46±8% during WBH (p>0.452).

Procedures

Height, body mass and skinfold thickness (biceps, triceps, supra-iliac and subscapular) were assessed on the first visit [24]. Thereafter, participants applied a rectal temperature probe 10 cm beyond the anal sphincter for the measurement of rectal temperature (Trec). A zero-heat flux temperature sensor (Bair Hugger, 3M, Minnesota, USA) was placed on the skin at the muscle belly of the vastus lateralis to measure deep tissue temperature (Tdt) [25]. The sensor was covered by a small Tupperware box to limit any thermal effects of the water-perfused suit. Tympanic temperature was measured by a temperature sensor (Squirrel, Grant Instruments, Shepreth, UK) worn throughout the session, secured in the left ear by cotton wool and industrial headphones. Nude body mass was then assessed to the nearest 0.1 kg (Seca 284, Hamburg, Germany). Skin thermometers were fitted on the chest, triceps, thigh and calf (Squirrel, Grant Instruments, Shepreth, UK), and a cannula was inserted into an antecubital vein. Participants rested in a supine position for 60 min wearing shorts and a T-shirt. At the end of the rest period, temperature measures, heart rate (HR; Polar, Kempele, Finland) and arterial blood pressure were recorded. Blood pressure was measured in duplicate using an automated cuff (Microlife, Cambridge, UK) at the brachial artery in the left arm. Thereafter, participants put on the water-perfused suit for the experimental condition. Physiological measures were assessed every 15 min, and at 30 min and 60 min post-session. After removing the water-perfused suit, a blood sample was collected, and the participant remained supine for an additional 30 min. Thereafter, nude body mass was measured. The final blood sample was collected 60 min post-session.

Participants were provided with water during the sessions to offset weight loss through sweating. During WBH, 150 ml of water was provided prior to and at 15 min intervals during heating. For LBH, 100 ml water was provided before and at the end of the heating protocol, while during CON 50 ml was provided directly following the 90 min session. Heart rate and systolic blood pressure were used to calculate rate pressure product as a measure of cardiac strain [26]; heart rate and Trec were used to calculate the physiological strain index [27].

Ultrasonography

Brachial and common femoral artery blood flow as well as cardiac output were assessed pre and directly post-heating as described in Hoekstra et al. [21]. Briefly, arterial blood flow and shear rate were assessed by ultrasonography (GE Healthcare, Chicago IL, USA) in duplicate at each time point and in the Doppler mode, which records arterial images and blood velocity signals simultaneously. Non-blinded measurements and analyses were performed by the same experienced ultrasonographer (T.O., 20 years of experience in ultrasonography), with a CV of 3.7% for femoral artery blood flow and 5.4% for brachial artery blood flow based on the baseline data obtained in CON. A16 MHz linear array transducer was used, and images were acquired at an insonation angle of 60° for 10 heart cycles. Arterial diameter was measured by identifying the adventitial border of the near and far walls of the artery using the built-in caliper function on the ultrasound unit. Two caliper measurements per image were taken and averaged to yield a diameter value. Blood flow was calculated as the product of the mean blood velocity during a cardiac cycle and the cross-sectional area of the vessel. Shear rate was calculated by the following formula: [4*(mean blood velocity/vessel diameter)] [28]. Vascular conductance in the femoral artery was determined as femoral artery blood flow/mean arterial pressure; the latter calculated as [(systolic blood pressure + (diastolic blood pressure*2))/3] [29].

Stroke volume and cardiac output were also measured by Doppler ultrasound (GE Healthcare, Chicago IL, USA), via the Doppler method [30]. Using a M5S transducer and keeping the participant in the left lateral decubitus position, left ventricular outflow tract diameter was measured using a parasternal long-axis view, while left ventricular flow (velocity time integral) was acquired in the 3- or 5- chamber view obtained immediately proximal to the aortic valve. These two variables were then used to calculate stroke volume. All cardiac measurements and analyses were performed by the same unblinded ultrasonographer (T.O.). Cardiac output was obtained as the product of stroke volume and HR.

Blood analyses

Blood was drawn into a K3EDTA and serum monovette. Plasma samples were centrifuged immediately for 10 min at 2360 g and 4°C. Serum samples underwent the same centrifugation procedure after they were allowed to clot for 30 min at room temperature. Plasma and serum aliquots were stored at -80°C until batch analysis. Enzyme-linked immunosorbent assays were used to determine serum and plasma BDNF concentration (R&D systems, Abingdon, UK) as well as plasma adrenaline concentration (Tecan UK Ltd, Reading, UK). Serum samples were diluted 30-fold. Haemoglobin concentrations and whole blood counts were assessed by a Yumizen H500 (Horiba Medical, Montpellier, France) automated analyser. Haematocrit, determined in duplicate using a microcentrifuge, and haemoglobin were used to correct BDNF concentrations and heamatological parameters for changes in plasma and blood volume, respectively [31]. The BDNF content in platelets was calculated according to the method postulated by Lommatszsch et al. [32]: [(serum BDNF concentration–plasma BDNF concentration)/platelet count].

Statistical analysis

All data are presented as mean ± SD. Normality and sphericity were checked by the Shapiro Wilk and Mauchley`s test, respectively. Changes in physiological, thermoregulatory and BDNF data were analysed by 2-way repeated measures ANOVA, with Fisher`s LSD tests used for post-hoc comparisons [33]. Data of WBH were used to calculate Pearson`s correlations between the change in serum, plasma and platelet BDNF, and Tcore, femoral artery shear rate and HR were calculated. The 24th version of SPSS (Chicago IL, USA) was used for all analyses and significance was accepted at p<0.05.

Results

Thermoregulatory measures

The thermoregulatory responses to the three experimental conditions are shown in Table 1. Rectal temperature at the end of WBH was 38.6±0.4°C, while its rise was reduced by upper-body cooling (Trec end LBH: 37.1±0.3°C; time x condition p<0.001). Nevertheless, Trec at the end of LBH was higher than CON (Trec end CON: 36.7±0.2°C; p = 0.001). There was an effect of time (p<0.001) and time x condition interaction (p<0.001) for tympanic temperature, with higher values in WBH compared with the other conditions throughout the session (p<0.006). There was no difference in tympanic temperature between LBH and CON at any time point (p>0.123). Deep tissue temperature was elevated to a larger extent by WBH compared with the other conditions (WBH: from 35.4±0.68 to 38.7±0.48, LBH: from 35.7±0.62 to 37.3±0.42, CON: from 35.3±0.36 to 36.1±0.24; time x condition p<0.001), while Tdt during LBH was also higher compared with CON from 30 min onwards (p<0.001). There was no difference in the change in body mass between conditions (CON: 0.08±0.07 kg, LBH: 0.07±0.21 kg, WBH: 0.18±0.32 kg; p = 0.321).

Table 1

Thermoregulatory responses to the three experimental conditions.

Values are expressed as mean ± SD (N = 10).

Parameter	Condition			Time
		Pre	45 min	End	P+30 min
Trec (°C)^	CON	36.6±0.4	36.6±0.2	36.7±0.2	36.7±0.2
	LBH	36.7±0.2	36.9±0.3	37.1±0.3*	36.5±0.3
	WBH	36.8±0.3	37.4±0.3#	38.6±0.4#	37.3±0.2
Ttympanic (°C)^	CON	35.4±0.4	35.7±0.4	35.8±0.4	35.5±0.3
	LBH	35.1±0.6	35.7±0.6	35.8±0.7	34.9±0.5
	WBH	35.3±0.6	37.0±0.3#	38.2±0.4#	35.2±0.6
Tthigh (°C)^	CON	31.6±1.0	34.0±0.6	34.0±0.6	32.6±0.9
	LBH	31.5±1.0	37.8±1.6*	38.0±1.3*	33.3±1.3
	WBH	32.4±1.1	38.6±0.7*	39.3±0.8#	33.4±1.0
Tcalf (°C)^	CON	33.2±0.9	34.0±1.0	34.0±1.0	33.2±1.0
	LBH	33.1±0.9	38.2±1.1*	38.5±1.1*	35.4±1.0*
	WBH	33.4±0.9	38.7±1.9*	39.7±1.8*	35.4±1.6*
Tarm (°C)^	CON	32.1±0.9	34.3±0.7	34.6±0.6	33.4±0.9
	LBH	32.2±0.8	23.1±4.0*	21.2±3.4*	26.8±2.2*
	WBH	32.1±0.7	38.5±0.8#	39.3±0.9#	35.1±1.1#
Tchest (°C)^	CON	32.8±1.3	34.2±0.6	34.5±0.7	33.0±1.0
	LBH	32.4±0.8	24.5±3.0*	21.3±3.2*	28.7±2.0*
	WBH	32.8±1.0	37.4±1.0#	38.5±1.3#	33.4±2.8#

CON: control; LBH: lower-body heating with upper-body cooling; WBH: whole-body heating; Trec: rectal temperature; Ttympanic: tympanic temperature; Tthigh: thigh skin temperature; Tcalf: calf skin temperature; Tarm: arm skin temperature; Tchest: chest skin temperature; P: post.

^ time x condition interaction

* different from CON

# different from other two conditions (p<0.05).

Cardiovascular measures

Cardiovascular outcome measures in response to the three experimental conditions are shown in Table 2. Blood flow in the common femoral artery was elevated to a larger extent by WBH compared with the other conditions (time x condition p<0.001), while it was also higher during LBH compared with CON at 45 min and 90 min (p = 0.002 at both time points). Shear rate in the femoral artery was higher in WBH compared with LBH (p<0.001), and in LBH compared with CON at 45 min and 90 min (p<0.038). There was an effect of time (p<0.001), condition (p<0.001) and a time x condition interaction (p<0.001) for rate pressure product, such that this was higher throughout WBH compared with the other conditions (p<0.001), while there was no difference between LBH and CON (p>0.127). An effect of time (p<0.001), condition (p<0.001) and a time x condition interaction (p<0.001) was present for the physiological strain index. The physiological strain index was higher throughout WBH compared with both other conditions (p<0.001), while it was also higher in LBH compared with CON at 45 min and 90 min (p<0.006).

Table 2

Cardiovascular responses to the three experimental conditions.

Values are expressed as mean ± SD (N = 10).

Parameter	Condition			Time
		Pre	45 min	End	P+60 min
HR (bpm)^	CON	58±15	59±13	55±11	56±13
	LBH	54±11	61±10	59±10	55±10
	WBH	59±15	90±16#	110±15#	62±17
SBP (mmHg)^	CON	114±9	125±10	126±7	117±10
	LBH	120±7	130±13	133±11	134±6
	WBH	118±8	134±12#	145±19#	126±9
DBP (mmHg)^	CON	63±5	65±8	68±5	67±5
	LBH	67±4	71±7	73±8	70±5
	WBH	64±6	64±4	70±7	63±6
CO (L/min)^	CON	4.4±1.0	-	4.2±0.9	-
	LBH	4.1±0.8	-	4.4±1.1	-
	WBH	4.5±1.0	-	7.0±1.8#	-
Brachial BF (ml/min)^	CON	98±49	-	107±44	-
	LBH	111±83	-	60±28*	-
	WBH	125±88	-	563±183#	-
Femoral BF (ml/min)^	CON	612±226	649±243	660±222	-
	LBH	609±226	842±261*	943±349*	-
	WBH	584±263	1427±332#	1713±409#	-
Femoral SR (/s)^	CON	54.4±18.3	54.0±15.7	52.5±16.3	-
	LBH	49.2±14.2	65.1±21.5*	68.6±27.4*	-
	WBH	51.9±16.6	124.3±38.6#	140.4±36.9#	-
Femoral VC (U)^	CON	6.8±2.1	7.0±1.8	6.9±1.9	-
	LBH	6.5±1.7	8.5±2.3*	9.6±4.0*	-
	WBH	6.2±2.1	15.5±2.4#	17.4±3.6#	-
RPP (AU)^	CON	6819±2328	7241±1942	7368±1610	6747±1417
	LBH	6937±1645	7774±1322	7903±1060	7092±1263
	WBH	7194±2286	11946±2624#	16403±3053#	8172±2628#
PSI (AU)^	CON	-	0.04±0.18	0.02±0.48	0.04±0.38
	LBH	-	0.93±0.73*	1.32±0.71*	0.05±0.61
	WBH	-	2.79±1.06#	5.66±1.17#	1.71±0.73#

CON: control; LBH: lower-body heating with upper-body cooling; WBH: whole-body heating; HR: heart rate; SBP: systolic blood pressure; DBP: diastolic blood pressure; CO: cardiac output; BF: blood flow; VC: vascular conductance; SR: shear rate; RPP: rate pressure product; PSI: physiological strain index; P: post.

^ time x condition interaction

* different from CON

# different from other two conditions (p<0.05).

Haematological measures

The haematological measures and adrenaline concentrations are shown in Table 3. An effect of time (p<0.001) and time x condition (p<0.001) but not condition (p = 0.277) was found for the platelet count. A higher platelet count was found immediately after WBH compared with the other conditions (p<0.002), while there was no difference between LBH and CON immediately post (p = 0.436). There was an effect of time (p = 0.006), but not of condition (p = 0.084) or time x condition (p = 0.866) for the total leukocyte count. Similarly, an effect of time was observed for monocytes (p = 0.026), but no effect of condition (p = 0.645) or a time x condition interaction (p = 0.669). For lymphocytes, there was an effect of time (p = 0.001) and a time x condition interaction (p = 0.001). At 60 min post-session, lymphocyte concentration was lower after WBH compared with the other two conditions (p<0.001). There was no effect of time (p = 0.060), condition (p = 0.074), nor a time x condition interaction effect (p = 0.809) for neutrophils. Plasma adrenaline concentration was higher following WBH compared with both other conditions (p<0.001) and following LBH compared with CON (p = 0.027).

Table 3

Adrenaline and haematological variables in responses to the three experimental conditions.

Values are expressed as mean ± SD (N = 10).

Parameter	Condition	Pre	Post	P+60 min
Adrenaline (ng/mL)$^	CON	12.2±7.9	16.0±10.0	19.3±14.0
	LBH	20.7±11.6	38.9±26.2*	33.0±19.7
	WBH	15.6±7.5	70.9±30.5#	13.8±7.3
Leukocytes (109/L)$	CON	4.54±1.03	4.96±1.15	4.77±0.92
	LBH	4.79±0.77	5.29±0.85	5.11±0.81
	WBH	5.16±1.35	5.99±1.63	5.62±1.87
Neutrophils (109/L)	CON	2.41±0.67	2.65±0.87	2.65±0.75
	LBH	2.68±0.57	2.69±0.55	2.94±0.62
	WBH	3.09±1.35	3.35±1.35	3.67±1.51
Monocytes (109/L)$	CON	0.40±0.14	0.42±0.13	0.39±0.11
	LBH	0.40±0.13	0.44±0.13	0.39±0.13
	WBH	0.40±0.12	0.46±0.15	0.42±0.17
Lymphocytes (109/L)$^	CON	1.49±0.33	1.61±0.34	1.50±0.25
	LBH	1.44±0.25	1.67±0.37	1.54±0.25
	WBH	1.46±0.38	1.37±0.75#	1.27±0.29#
Platelets (109/L)$^	CON	199±20	205±25	207±22
	LBH	209±33	211±31	209±38
	WBH	189±27	235±19#	197±36
Δ Plasma volume (-fold)^	CON	N/A	1.06±0.11#	1.05±0.06#
	LBH	N/A	0.94±0.07	0.95±0.06
	WBH	N/A	0.92±0.06	0.95±0.08

Abbreviations: CON: control; LBH: lower-body heating with upper-body cooling; WBH: whole-body heating.

$ effect of time

^ time x condition interaction

* different from CON

# different from other two conditions (p<0.05).

Brain-derived neurotrophic factor

The acute changes in serum and plasma BDNF concentration and BDNF release per platelet following the three experimental conditions are shown in Fig 1. There was no effect of time (p = 0.116) or condition (p = 0.145) for serum BDNF. However, there was a time x condition interaction effect (p = 0.033). Directly post-session, serum BDNF concentration was increased in WBH (p = 0.044), but not LBH (p = 0.126) or CON (p = 0.454). Serum BDNF concentration immediately after the session was higher in WBH and LBH compared with CON (p = 0.048), with no difference between WBH and LBH (p = 0.206). Plasma BDNF concentration showed an effect of time (p<0.001) and condition (p = 0.022), as well as a time x condition interaction effect (p = 0.001). Immediately following the session, plasma BDNF concentration was increased in WBH (p = 0.003), but not LBH (p = 0.468) or CON (p = 0.053). There was a difference between WBH and CON (p = 0.009), but not between LBH and CON immediately after the session (p = 0.134). Finally, no effect of time (p = 0.392), condition (p = 0.220), or time x condition interaction (p = 0.428) was found for the BDNF content per platelet.

Fig 1

Brain-derived neurotrophic factor responses in serum and plasma following the three experimental conditions.

* Different from Pre, ^ different from CON (p<0.05).

Correlations

There was a strong, positive correlation between the change in serum BDNF concentration and BDNF per platelet (r = 0.90, p<0.001). There was no correlation between the change in serum BDNF concentration and plasma BDNF concentration (r = 0.43, p = 0.24), platelet count (r = 0.07, p = 0.861) or the physiological measures (ΔTrec r = 0.06, p = 0.846; Δfemoral artery shear rate r = -0.16, p = 0.652, ΔHR r = -0.15, p = 0.689). The acute change in plasma BDNF concentration was not correlated with the change in platelet BDNF (r = 0.05, p = 0.900), platelet count (r = 0.47, p = 0.203) or any of the physiological measures (ΔTrec r = 0.14, p = 0.720; Δfemoral artery shear rate r = 0.07, p = 0.861, ΔHR r = 0.17, p = 0.665). Platelet BDNF was not correlated with platelet count (r = -0.31, p = 0.379) or any of the physiological measures (ΔTrec r = -0.07, p = 0.841; Δfemoral artery shear rate r = 0.02, p = 0.941, ΔHR r = -0.36, p = 0.309).

Discussion

This study investigated the efficacy of WBH as well as a local heating protocol to induce an acute BDNF response. Upper-body cooling during passive heating reduced cardiac output, the rate pressure product as well as the physiological strain index when compared with WBH. However, while WBH acutely elevated plasma and serum BDNF concentration, this response was blunted after LBH, despite an increase in sympathetic activity and femoral artery shear rate.

Passively elevating body temperature by WBH induced an acute increase in circulating BDNF, corroborating the findings from Kojima et al. [11], who reported an increase in serum BDNF concentration following 20 min of hot water immersion. The effect of an elevated body temperature on peripheral BDNF may be mediated by a range of factors acting on multiple cellular sources of BDNF. For instance, exercising in a warm environment has been shown to increase S100β, a marker for blood-brain-barrier permeability [34], which may increase the contribution of the brain to plasma BDNF [17]. Conversely, Kojima et al. [11] found significant increases in serum BDNF concentration without concomitant changes in S100β following 20 min of hot water immersion. In support, Shepley et al. [35] recently reported that 60 min of moderate-to-severe hyperthermia induced by hot water immersion (+2°C core temperature) has a negligible impact on biomarkers of neurovascular integrity and permeability released from the brain. This highlights the fact that sources other than the brain produce and release BDNF in response to various physiological stimuli [17].

As an example of such a physiological stimulus, the vascular endothelium produces and secretes BDNF in response to shear stress in vitro [19], and the expression of the BDNF-receptor TrkB on endothelial cells has led others to suggest that a positive feedback loop exists in which the binding of circulating BDNF with TrkB activates BDNF production by endothelial cells [17]. WBH induced a ~3- and ~4-fold increase in femoral artery and brachial artery blood flow, respectively, potentially contributing to BDNF release by endothelial cells in the vasculature. Further, the increase in cardiac output and adrenaline indicates an increase in sympathetic activity during WBH. Walsh et al. [20] support the importance of sympathetic activity for the increase in circulating BDNF concentration. By investigating handgrip exercise, the authors exploited the notion that local metabolic stress within skeletal muscle during exercise appears more important than the absolute muscle mass involved for sympathetic activation [36]. Targeting sympathetic activity by this small muscle mass exercise increased serum BDNF concentration, despite the limited metabolic cost and body temperature rise associated with the activity. The effect of sympathetic activity on serum BDNF concentration may have been mediated by the recruitment of platelets from the spleen into the circulation that occurred during WBH, indicated by the increase in platelet count. However, contrary to this notion, there was no correlation between the change in platelet count and serum BDNF concentration following WBH. Indeed, the relationship between circulating platelets and BDNF is impacted by more than platelet count per se, as aspects of platelet function are altered by passive heating in humans [37] or in pathological states like major depressive disorder [38]. In addition, as for the lack of significant correlations between the BDNF response and measures of blood flow and sympathetic activity, it should be noted that this study included a relatively small sample size and was not designed to explore correlations between these outcome measures.

In contrast to the acute increase in serum and plasma BDNF concentration after WBH, limiting the rise in Trec by the localised cooling used in LBH blunted this response. In the exercise literature, there appears to be a dose-response relationship between exercise intensity and the acute increase in BDNF concentration [7, 39]. For instance, a systematic review showed that 69% of studies investigating a high-intensity exercise protocol reported an acute increase in BDNF concentration, while this was only 44% in studies on low and moderate-intensity exercise [39]. This could be explained by the larger increase in shear stress [40] and sympathetic activity [41] during high compared with moderate-intensity exercise. As the recently put forward exercise intensity threshold for the elevation of BDNF concentration [42] may thus be related to these factors, it is likely that the increase in shear stress and sympathetic activity by LBH was not sufficient to elevate BDNF concentration. Indeed, although LBH induced a small increase in adrenaline concentration, none of the other measures of sympathetic activity were elevated (e.g. cardiac output, diastolic blood pressure); despite the large increase in Tskin of the lower limbs. This underscores the relatively large contribution of Tcore when compared with Tskin to sympathetic activity-related processes such as changes in vasomotor activity and catecholamine production [43]. In line with the limited increase in sympathetic activity, LBH resulted in a 40% rise in shear rate compared with nearly 300% in WBH. Future attempts to create a tolerable passive heating protocols to elevate circulating BDNF concentration may thus need to induce a larger increase in Tcore; of which the exact magnitude will depend on the balance between the attendant cardiovascular strain and the acute BDNF response. As such, heating the lower limbs in the absence of upper-body cooling may be an appealing protocol to test in future studies.

In the present study, BDNF concentration after WBH was elevated in serum as well as plasma. This suggests that passively elevating body temperature stimulates BDNF release by peripheral tissues (primarily reflected by plasma measurements) as well as platelets (primarily reflected by serum measurements). Interestingly, in contrast to Kojima et al. [11], the increase in serum BDNF concentration found after WBH in the present study was accompanied by an elevated platelet count. Although the lack of BDNF mRNA expression in platelets suggest that these cells do not synthesise BDNF de novo [44], BDNF stored in platelets is released during the clotting process of the serum collection procedure [45]. In vitro experiments suggest that shear stress and sympathetic activation can enhance BDNF release per platelet [18]. However, there was no change in BDNF release per platelet following WBH, suggesting that the elevated serum BDNF concentration in the present study was mainly the result of an increase in platelet count. On the other hand, despite no change in BDNF release per platelet in WBH, there was a strong correlation (r = 0.90) between the change in serum BDNF concentration and BDNF per platelet. Regardless, it should be noted that the calculated BDNF per platelet is derived from an indirect method with inbuilt assumptions about the sources of BDNF in plasma and serum [32]. Future studies could employ direct biochemistry techniques to assess BDNF content and release by platelets in response to physiological stress to further investigate the role of platelets in the concentration of circulating BDNF [37, 38].

Practical applications and future directions

The acute elevation of BDNF concentration following WBH provides strong rationale to further investigate the efficacy of passive heating protocols to elevate BDNF concentration and improve cognitive function. At the same time, whilst more research on the safety of heat therapy is warranted, it should be noted that whole-body passive heat stress may be contraindicated for some individuals. For example, an observational report noted the occurrence of a cardiac arrest during hot water bathing in 9.84 out of 100,000 people, while the level of consciousness after an adverse event in the bath was negatively related to the core temperature attained [46]. In addition, whole-body heating is associated with higher thermal discomfort and more negative affective responses compared with local heating [21]. As such, to confidently prescribe effective and safe passive heating protocols for a wide range of (clinical) populations (e.g., older adults and persons with chronic heart failure), further research into passive heating protocols with a reduced cardiovascular strain and thermal discomfort is needed. Aside from exploring additional tolerable and yet effective protocols, chronic intervention studies could build on Glazachev et al. [12] to further investigate the effects of repeated passive heating on BDNF concentration and cognitive function. Importantly, such studies should focus on persons at risk for reduced BDNF expression or cognitive function due to metabolic dysfunction or impaired mobility. While such individuals may arguably benefit most from interventions that elevate BDNF concentration, physiological responses to heat stress can be impacted by old age [15] and health conditions such as type II diabetes mellitus [16]; reinforcing the need for studies in specific populations.

In conclusion, while the local cooling applied in LBH reduces cardiovascular strain when compared with WBH, this protocol does not elevate circulating BDNF concentration. A larger increase in shear rate and sympathetic activity, potentially through elevating Tcore to a larger extent than in LBH, may thus be needed to induce an acute BDNF response through passive heating. In contrast, the acute increase in plasma and serum BDNF concentration following WBH provides further support to pursue research into the potential of passive heat therapy to elevate circulating BDNF concentration.

18 Jun 2021

PONE-D-21-13550

Serum and plasma brain-derived neurotrophic factor concentration are elevated by systemic but not local passive heating

PLOS ONE

Dear Dr. Hoekstra,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please respond to the reviewers comments with detailed responses.

Please submit your revised manuscript by Jul 31 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Caroline Sunderland

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for submitting the above manuscript to PLOS ONE. During our internal evaluation of the manuscript, we found significant text overlap between your submission and the following previously published works, some of which you are an author.

https://cdnsciencepub.com/doi/10.1139/apnm-2020-0704

We would like to make you aware that copying extracts from previous publications, especially outside the methods section, word-for-word is unacceptable. In addition, the reproduction of text from published reports has implications for the copyright that may apply to the publications.

https://cdnsciencepub.com/doi/10.1139/apnm-2020-0704

Please revise the manuscript to rephrase the duplicated text, cite your sources, and provide details as to how the current manuscript advances on previous work. Please note that further consideration is dependent on the submission of a manuscript that addresses these concerns about the overlap in text with published work.

We will carefully review your manuscript upon resubmission, so please ensure that your revision is thorough.

3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

Additional Editor Comments:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This study evaluates the response of circulatory BDNF to different heating strategies, all using a water perfused suit but targeting different areas of the body. Findings suggest the impact of whole body heating on circulating BDNF concentration is greater in comparison to lower body heating alone, highlighting the need for greater internal heat alterations to influence BDNF. Rectal temperature was greater in the lower body heating protocol when compared to the control, however this was not great enough to influence BDNF.

This study generates a strong rational for the need to know the impact of passive heat stress, based on the assumption this would be a better option for improving health outcomes for those populations unable exercise, a well supported method of improving BDNF responses. This study adds to the current literature where local heating strategies have not previously been assessed in relation to BDNF.

Although this study highlights a strong rational around the benefits for use in populations who can’t exercise, it does not detail any of the potential side effects/negative implications. Having an understanding of how the target population’s discomfort and affect is influenced may provide greater application and understanding when the research proceeds to use it in the desired population.

There are no major issues with this study and it’s design. However, some minor recommendations detailed below:

Specifics:

An excellent overview of BDNF and it’s various effects are detailed. An extensive mechanistic approach with complex measurements are included in the study.

Providing a more in depth evaluation of the populations this strategy applies to would strengthen your rational.

Dinooff et al (2016) – what types of exercise? In humans? Elaborate.

General – add specifics around types of exercise and heat stress used for literature within the introduction.

Good rational provided based on why other protocols are not applicable.

Methods would benefit from more detail on water perfused suit, specifically how the upper body cooling/lower body heating protocol was implemented. Including a figure (supplementary material) would be beneficial.

Upper body cooling is used in the local heating strategy – what implications does this have compared to if a thermoneutral option was used there. Worth discussing why thermoneutral was not used in upper body with lower body heating. (This may be better understood when methods of the suit are elaborated on).

Were individual differences in sweat rate accounted for? What is the rational for the drink volume used?

Were any discomfort measures or measures of affect taken? Practical application – is it feasible to use in elderly, disabled or individuals with disease without knowledge of the levels of discomfort and the influence on feeling/affect in a healthy population.

Shear stress is discussed throughout when referring to the potential mechanisms involved – this discussion could be strengthened through a more in depth introduction to this earlier in the paper.

All tables and figures are relevant and well formatted.

Reviewer #2: The manuscript by Ogawa et. al. titled, “Serum and plasma brain-derived neurotrophic factor concentration are elevated by systemic by not local passive heating” determines the impact of whole body vs lower body heating on BDNF and cardiovascular parameters. This study appears to be carefully conducted and prepared. I offer the following comments for the authors to consider.

Abstract:

• I know space is limed, but the abstract would be enhanced by including some of the actual data instead of just p-values. Perhaps at least for some of you main dependent variables.

Introduction:

• In the second paragraph, second sentence: I believe you mean is and not in (…especially in situations where exercise is not accessible….

• I believe there is some literature that indicates no difference in the exercise response between different environmental temperatures (Collins et al, 2017, Temperature). I just wonder if this would be useful here or in the discussion to help set up the notion that the BDNF response is likely more related to cardiovascular strain than the temperature??? Don’t feel obligated on this comment as I think it is adequately presented as is, but it may be worth considering??

Methods:

• For the food diary, was the diet of the first trial replicated in the other two. Please provide some info on how this was used.

• Why was the CON at 36°C? Throughout the manuscript, I don’t really like the word thermoneutral. Is a 36°C skin temperature what you consider thermoneutral. I was finding thermoneutral skin temp to be 33-35 and other things indicating 28-32 air temperature? Perhaps some justification/references are needed here. I fear that thermoneutral may mean different things to different people and the working should be changed or justified. I don’t have a problem with the use of 36 C, just perhaps tweaking the presentation.

• You report the temp and humidity of the lab. I commend you for adding this detail. Perhaps add a p-value from statistical analysis so that the reader can more readily and quickly interpret this info.

• I was having a hard time finding your temperature sensors based on the given information. Please provide a product # or name? Is the zero-heat flux temp senor validated for this use and is there a reference to include? Was there a specific data logger that you used? I was just trying to picture the exact set-up and was having a hard time. I think that by adding more detail here it would enhance this section.

• How did you determine the water volume provided? It doesn’t appear to be based on the actual weight change of the subject?

• Statistics: I would suggest using “Fishers Protected LSD method” verbiage for describing your post-hoc. It is exactly what you did with the t-test, but the verbiage helps remind the reader that your error rate is accounted within the ANOVA itself. Not a big deal, just something that I find helps if others (reviewers or readers) criticize this approach.

Discussion:

• My interpretation of the discussion is that the BDNF response is not directly related to the heat, but rather indirectly via the cardiovascular stress which is supported by the data. This is a bit difficult based on the lack of correlation between BDNF and cardiovascular stress parameters. Could skin thermal receptors play a role here by activating the sympathetic nervous system. The thought here would be that the WBH activates more of the receptors than the LBH and lead to the differences? This comment is probably beyond the scope of your project, but may be something to consider as you think about the potential mechanism in light of the lack of correlations with some of the cardiovascular correlations. I think it is important to further discuss these lack of correlations. I just kept thinking of technical issues around the use of correlations (relatively low sample size, having 3 temperature conditions instead a larger range, ect.) and what other factors may stimulate this alteration. I wouldn’t do too much here, but you may want to address the correlations a bit more.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

14 Jul 2021

Dear Reviewer 1,

Thank you for the careful reading of our manuscript and the insightful comments. We also appreciate your comment regarding the potentially negative impact of (whole-body) passive heating for certain populations. We have tried to incorporate your suggestions and believe they have improved the quality of the manuscript.

This study evaluates the response of circulatory BDNF to different heating strategies, all using a water perfused suit but targeting different areas of the body. Findings suggest the impact of whole body heating on circulating BDNF concentration is greater in comparison to lower body heating alone, highlighting the need for greater internal heat alterations to influence BDNF. Rectal temperature was greater in the lower body heating protocol when compared to the control, however this was not great enough to influence BDNF. This study generates a strong rational for the need to know the impact of passive heat stress, based on the assumption this would be a better option for improving health outcomes for those populations unable exercise, a well supported method of improving BDNF responses. This study adds to the current literature where local heating strategies have not previously been assessed in relation to BDNF.

Thank you for the kind words.

Although this study highlights a strong rational around the benefits for use in populations who can’t exercise, it does not detail any of the potential side effects/negative implications. Having an understanding of how the target population’s discomfort and affect is influenced may provide greater application and understanding when the research proceeds to use it in the desired population.

We agree that this is an important consideration. This aligns closely to other comments by you and the other reviewer. As such, we have incorporated discussion on the implications for specific populations and the potential thermal discomfort during whole-body heating in a “practical application and future directions” paragraph at the end of the Discussion.

There are no major issues with this study and it’s design. However, some minor recommendations detailed below:

Specifics:

An excellent overview of BDNF and it’s various effects are detailed. An extensive mechanistic approach with complex measurements are included in the study.

Thank you for the kind words on the Introduction.

Providing a more in depth evaluation of the populations this strategy applies to would strengthen your rational.

Thank you for this comment.

We agree that this is an important consideration. We have now added a sentence on the potential implications of an effective, but more tolerable passive heating protocol in the Introduction (Line 95). As highlighted in the Introduction, this can make this intervention more accessible to people with chronic heart failure and older adults for example. Additionally, as we have previously shown that a local heating protocol can lead to more favourable perceptual responses (PMID: 33439769), we believe the implications of such a protocol may extend to all individuals using heat therapy for health promotion. As mentioned above, these considerations are now also included in the Discussion (Line 363).

Dinooff et al (2016) – what types of exercise? In humans? Elaborate.

We have now made it clear that this is primarily aerobic exercise, and that Dinoff et al (2016) has provided evidence for this effect in humans. We have kept it brief, as passive heat stress was the primary focus of the manuscript (see also the comment below).

General – add specifics around types of exercise and heat stress used for literature within the introduction.

We have now added specifics related to the heat stress described, as this is the focus of the manuscript (e.g. Line 74, Line 292).

Good rational provided based on why other protocols are not applicable.

Thank you.

Methods would benefit from more detail on water perfused suit, specifically how the upper body cooling/lower body heating protocol was implemented. Including a figure (supplementary material) would be beneficial.

Thank you for this comment and we agree that this suggestion would clarify the intervention for the reader. We have published on other outcome measures in response to the protocol employed in the current study and have now more clearly referred the reader to that publication (Line 122; PMID: 33439769), as a detailed outline has been provided in that article.

Upper body cooling is used in the local heating strategy – what implications does this have compared to if a thermoneutral option was used there. Worth discussing why thermoneutral was not used in upper body with lower body heating. (This may be better understood when methods of the suit are elaborated on).

The reason for the upper-body cooling instead of a thermoneutral upper body condition was that we wanted to investigate a condition in which core temperature rises were limited, whilst a local heat stress was applied (a more substantial rise in core temperature was likely to occur with heating of the legs in combination with a thermoneutral condition for the upper body (PMID: 30303416)). Nonetheless, considering the absence of an acute BDNF response after the LBH protocol in the current study, the suggested combination of heating the legs without cooling the upper body would be an interesting protocol to investigate in the future.

Were individual differences in sweat rate accounted for? What is the rational for the drink volume used?

The water volume provided during the sessions was based on pilot work. We have not taken into account individual differences in sweat rate, and agree that this would have been a good addition to the study. However, considering the lack of difference in body mass loss and plasma volume change between the conditions, we believe that the potential effect of hydration status on BDNF concentration (PMID: 28828079) was likely to have been negligible in the current study.

Were any discomfort measures or measures of affect taken? Practical application – is it feasible to use in elderly, disabled or individuals with disease without knowledge of the levels of discomfort and the influence on feeling/affect in a healthy population.

Thank you for this relevant comment. As mentioned previously, we have published on other outcome measures in response to the same experimental protocols. The perceptual responses to the heating protocols were one of the main outcome measures of that article (PMID: 33439769), including measures of affect, thermal comfort and thermal sensation. We have included discussion and a reference to this article in the newly added “practical implications and future directions” paragraph of the Discussion.

Shear stress is discussed throughout when referring to the potential mechanisms involved – this discussion could be strengthened through a more in depth introduction to this earlier in the paper.

We have now included discussion on shear stress as a potential stimulator of BDNF production and how it provides rationale to investigate local passive heating protocols in the Introduction section (Line 95).

All tables and figures are relevant and well formatted.

Thank you.

Dear Reviewer 2,

Thank you for your insightful comments and suggestions to improve the quality of the manuscript. We are also grateful for your valuable suggestions as to what the mechanisms underpinning the BDNF response to heat stress may be. We believe that the changes we have made based on your comments have improved the quality of the manuscript.

The manuscript by Ogawa et. al. titled, “Serum and plasma brain-derived neurotrophic factor concentration are elevated by systemic by not local passive heating” determines the impact of whole body vs lower body heating on BDNF and cardiovascular parameters. This study appears to be carefully conducted and prepared. I offer the following comments for the authors to consider.

Abstract:

• I know space is limed, but the abstract would be enhanced by including some of the actual data instead of just p-values. Perhaps at least for some of you main dependent variables.

Thank you for this comment and we agree that this strengthens the abstract. We have now included the serum and plasma BDNF data.

Introduction:

• In the second paragraph, second sentence: I believe you mean is and not in (…especially in situations where exercise is not accessible….

We have now changed in to is.

• I believe there is some literature that indicates no difference in the exercise response between different environmental temperatures (Collins et al, 2017, Temperature). I just wonder if this would be useful here or in the discussion to help set up the notion that the BDNF response is likely more related to cardiovascular strain than the temperature??? Don’t feel obligated on this comment as I think it is adequately presented as is, but it may be worth considering??

This is an interesting suggestion. Indeed, one could argue that as long as for instance shear stress is elevated (through exercise, for example), an additional increase in temperature may not be necessary to increase BDNF concentration. However, the lack of such cardiovascular measurements in the exercise studies (Collings et al. 2017; Goekint et al., 2011) makes it difficult to uncouple the effect of hyperthermia and e.g. shear stress with any confidence. Moreover, there is evidence for the contrary (i.e. larger BDNF response in hot environment (Goekint et al., 2011; PMID: 21385602)), and closer inspection of the data presented in Collins et al. (2017) shows that BDNF concentration is ~25% higher after exercise in the heat compared with the cold and moderate conditions; suggesting that the lack of effect in that study may have been related to limited statistical power. Therefore, also for the sake of clarity in the Introduction, we prefer to not include Collins et al. (2017) in this section. However, we agree with your interpretation that the BDNF response is likely closer related to factors such as shear stress and sympathetic activation than hyperthermia per se, and have tried to emphasise that further based on your comments (e.g. Line 95 - 98).

Methods:

• For the food diary, was the diet of the first trial replicated in the other two. Please provide some info on how this was used.

Thank you for this comment. We have now described this process in more detail (Line 121).

• Why was the CON at 36°C? Throughout the manuscript, I don’t really like the word thermoneutral. Is a 36°C skin temperature what you consider thermoneutral. I was finding thermoneutral skin temp to be 33-35 and other things indicating 28-32 air temperature? Perhaps some justification/references are needed here. I fear that thermoneutral may mean different things to different people and the working should be changed or justified. I don’t have a problem with the use of 36 C, just perhaps tweaking the presentation.

Thank you for this comment. We used this condition as water-perfused suit temperatures in the range of 33-36°C can keep core temperature stable (PMID: 30303416; PMID: 16763078). Pilot work in preparation for this study showed that water-perfused temperatures below 36°C resulted in a core temperature reduction. As such, this temperature was chosen as the control condition.

Despite this rationale, we agree that this is not truly thermoneutral, as the suit temperature was substantially higher than resting skin temperature. Therefore, we have removed the term thermoneutral and have changed this to “control condition” throughout the manuscript.

• You report the temp and humidity of the lab. I commend you for adding this detail. Perhaps add a p-value from statistical analysis so that the reader can more readily and quickly interpret this info.

Thank you for this comment. We have now included the smallest p-value of the one-way ANOVAs conducted on the comparisons for temperature and humidity (p>0.452).

• I was having a hard time finding your temperature sensors based on the given information. Please provide a product # or name? Is the zero-heat flux temp senor validated for this use and is there a reference to include? Was there a specific data logger that you used? I was just trying to picture the exact set-up and was having a hard time. I think that by adding more detail here it would enhance this section.

Thank you for this comment. The Bair Hugger sensor is a coin-sized sensor that was placed on the vastus lateralis and covered with a small Tupperware box. The sensor has been validated previously, and we have now included a reference for this method (Binzoni et al., 1999). We hope that this reference, together with the reference to the product and manufacturer already provided, will give the reader sufficient information to visualise and evaluate this outcome measure.

More generally, we have now clearer referred the reader to a manuscript we have previously published, describing a different set of outcome measures with the same experimental procedures (Hoekstra et al., 2021; APNM). Here the experimental set-up is described in more detail, and the readers can refer to this article if more specific information is required. This article also includes a detailed image of the experimental set-up.

• How did you determine the water volume provided? It doesn’t appear to be based on the actual weight change of the subject?

Thank you for this comment. The water provided was based on the mean body mass loss that we observed in several participants (N=4) during our pilot testing. We agree that we did not assess individual sweat rate for each participant separately, and thus we did not achieve exact euhydration in each trial. However, considering the lack of statistical difference in body mass loss between trials, we believe we have managed to avoid the potentially confounding effect of dehydration in the present study.

• Statistics: I would suggest using “Fishers Protected LSD method” verbiage for describing your post-hoc. It is exactly what you did with the t-test, but the verbiage helps remind the reader that your error rate is accounted within the ANOVA itself. Not a big deal, just something that I find helps if others (reviewers or readers) criticize this approach.

Thank you for this suggestion. We have now changed this in the statistical analysis section.

Discussion:

• My interpretation of the discussion is that the BDNF response is not directly related to the heat, but rather indirectly via the cardiovascular stress which is supported by the data. This is a bit difficult based on the lack of correlation between BDNF and cardiovascular stress parameters. Could skin thermal receptors play a role here by activating the sympathetic nervous system. The thought here would be that the WBH activates more of the receptors than the LBH and lead to the differences? This comment is probably beyond the scope of your project, but may be something to consider as you think about the potential mechanism in light of the lack of correlations with some of the cardiovascular correlations. I think it is important to further discuss these lack of correlations. I just kept thinking of technical issues around the use of correlations (relatively low sample size, having 3 temperature conditions instead a larger range, ect.) and what other factors may stimulate this alteration. I wouldn’t do too much here, but you may want to address the correlations a bit more.

Thank you for your accurate interpretation of our discussion of the data; we agree that it is indeed likely that the BDNF response is more closely linked to some of the physiological consequences of heat stress than hyperthermia itself (see e.g. PMID: 12008958). This is something we have tried to lay out in the paragraph starting at Line 297. However, we agree that the lack of correlations between the BDNF response and such physiological markers is somewhat surprising. In line with your note of caution, we have now included a sentence on the limitations of the correlation analyses in the present study (Line 316).

Submitted filename: Response to Reviewers Ogawa et al.docx

Click here for additional data file.

17 Nov 2021

Serum and plasma brain-derived neurotrophic factor concentration are elevated by systemic but not local passive heating

PONE-D-21-13550R1

Dear Dr. Hoekstra,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Caroline Sunderland

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have made adequate amendments to the paper to warrant publication. Greater clarity has bee provided in specific areas and all comments have been addressed.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

1 Dec 2021

PONE-D-21-13550R1

Serum and plasma brain-derived neurotrophic factor concentration are elevated by systemic but not local passive heating

Dear Dr. Hoekstra:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Caroline Sunderland

Academic Editor

PLOS ONE