Chronic sympathectomy of the caudal artery delays cutaneous heat loss during passive heating

Neuroscience Letters 537 (2013) 11–16

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Chronic sympathectomy of the caudal artery delays cutaneous heat loss during passive heating Milene Rodrigues Malheiros Lima a , Washington Pires a , Ivana Alice Teixeira Fonseca a , Cletiana Gonc¸alves Fonseca a , Patrícia Massara Martinelli b , Samuel Penna Wanner a , Nilo Resende Viana Lima a,∗ a b

Exercise Physiology Laboratory, School of Physical Education, Physiotherapy and Occupational Therapy, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Department of Morphology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

h i g h l i g h t s     

Caudal artery denervation slows down cutaneous heat loss during heat exposure. Caudal artery denervation decreases the temperature variability of tail skin. The variability of the core temperature is not altered by caudal artery denervation. No signs of reinnervation were observed 30 days after caudal artery denervation. Sympathetic innervation provides fine-tuned control over cutaneous heat loss.

a r t i c l e

i n f o

Article history: Received 8 October 2012 Received in revised form 18 December 2012 Accepted 7 January 2013 Keywords: Ambient temperature Denervation Heat Hyperthermia Rat tail Skin temperature

a b s t r a c t The present study aimed to investigate the chronic effects of caudal artery sympathectomy on thermoregulatory adjustments induced by passive heating. Male Wistar rats were subjected to two surgical procedures: caudal artery denervation (CAD) or sham surgery (Sham-CAD) and intraperitoneal implantation of a temperature sensor. On the day of the experiments, the animals were exposed to an ambient temperature of 36 ◦ C for 60 min or allowed to rest under thermoneutral conditions (26 ◦ C). During the experiments, the tail skin temperature (Tskin ) and the core body temperature (Tcore ) were measured. Under thermoneutral conditions, although sympathetic denervation did not change the average values of Tcore and Tskin , CAD rats exhibited decreased Tskin variability compared with Sham-CAD rats (0.020 ± 0.005 ◦ C vs. 0.031 ± 0.005 ◦ C; P = 0.024). During heat exposure, no differences were observed in the Tcore between the groups. In contrast, although peak Tskin values were not affected by chronic sympathectomy of the caudal artery, CAD animals showed a delayed increase in Tskin ; the time until the stabilization of Tskin was three-fold longer in CAD rats than in Sham-CAD rats (15.3 ± 2.5 min vs. 4.9 ± 0.6 min; P = 0.001). In conclusion, chronic sympathectomy of the caudal artery delays cutaneous heat loss during passive heating and decreases Tskin variability under thermoneutral conditions. Taken together, our results indicate that the sympathetic innervation of cutaneous vessels is essential for the precise regulation of tail heat loss. © 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Some pathological conditions (e.g., spinal cord lesions or diabetic neuropathy) and sympathectomy of the upper limbs (used as a treatment for palmar hyperhidrosis) change the

∗ Corresponding author at: Av. Antônio Carlos, 6627 Pampulha, Belo Horizonte 31270-901, MG, Brazil. Tel.: +55 31 3409 2351. E-mail addresses: [email protected] (M.R.M. Lima), [email protected] (W. Pires), [email protected] (I.A.T. Fonseca), [email protected] (C.G. Fonseca), [email protected] (P.M. Martinelli), [email protected] (S.P. Wanner), [email protected] (N.R.V. Lima). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.01.013

perivascular neural environment and, consequently, the regulation of blood flow in the skin [2,9,12]. The sympathetic denervation of cutaneous vessels promotes a transitory increase in skin blood flow that is followed by a progressive and persistent decrease [23]; this late reduction of cutaneous blood flow has been related to functional and structural adaptations that favor greater constriction of the denervated vasculature [1,17,19,21,23]. Because the regeneration of the sympathetic axons innervating the distal vessels is slow and incomplete [3], lesions in the cutaneous vascular nerves can make the skin cold, cyanotic, and susceptible to injury (i.e., ulceration) [12,20]. Several methodological approaches, such as surgical procedures [7,23], pharmacological treatments [17] and studies in

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patients and animals with nerve injuries [4,12], have been performed to investigate both the acute and chronic adaptations induced by vascular denervation. Although these studies have enhanced the knowledge about the effects of sympathetic innervation, the results of such studies cannot be attributed solely to the lack of vascular innervation. In pathological models, the results are also associated with a loss of cutaneous sensorial innervation and/or vascular dysfunctions inherent to the diseases. Furthermore, the wide-spread side effects evoked by the systemic administration (i.e., intravenous or intraperitoneal administration) of pharmacological agents or the concomitant denervation of the vascular beds of the feet induced by lumbar sympathectomy surgery can also affect the experimental outcomes [6,7]. Therefore, to avoid the limitations of the experimental approaches employed so far, we developed a surgical procedure that allowed us to exclusively investigate the effects of sympathectomy on a specific blood vessel. For this purpose, a phenol solution was topically applied around the proximal portion of the caudal artery. The application of phenol induces fast and long-lasting nerve degeneration [22]. The cutaneous vessels in the rat’s tail play an important role in the control of heat exchange between the body surface and the environment. Cutaneous blood flow is primarily under the control of the sympathetic vasoconstrictor nerves [7], and in thermoneutral environments, core body temperature (Tcore ) regulation is largely dependent on nonevaporative physical processes that include fluctuations in skin blood flow [11]. Dysfunctions in thermoregulatory adjustments have been described in animals and human patients with spinal cord injuries, in individuals subjected to sympathectomy of the upper limbs and in patients with diabetic neuropathy [9,12]; however, because other mechanisms can interfere with these responses (lack of cutaneous sensorial innervation, lack of sweat gland innervation, and changes in temperature sensitivity), the exclusive contribution of sympathetic vascular innervation to body temperature control has yet to be conclusively determined. Taking these facts in account, the present study aimed to investigate whether the cutaneous heat loss of freely moving animals in a neutral environment is changed by chronic sympathectomy of the caudal artery. Kalincik et al. [4] reported that during cold exposure, the magnitude of the tail skin temperature (Tskin ) reduction was not changed in rats with spinal cord lesions, suggesting that a higher sensitivity to humoral and local vasoconstrictor agents could have compensated for the absence of neurovascular control in this environment. Alternatively, it is possible that vascular adaptations favoring vasoconstriction could impair cutaneous heat loss in warm environments, consequently increasing the risk of heatrelated lesions. However, no study has determined whether the thermoregulatory adjustments induced by heat exposure are modified by chronic sympathectomy of the caudal artery. This was the second objective of the present study.

2. Materials and methods All experimental procedures were approved by the Ethics Committee of the Universidade Federal de Minas Gerais for the Care and Use of Laboratory Animals (protocol 109/09) and were carried out in accordance with the policy described in the Committee’s Guiding Principles Manual. The experiments were performed on 26 male Wistar rats weighting 210–240 g at the time of sympathectomy. Animals were housed in individual cages and kept in a room with controlled light (05:00–19:00 h) and temperature (24.0 ± 1.0 ◦ C) conditions. Standard rat chow and tap water were provided ad libitum. Initially, the rats were subjected to caudal artery denervation (CAD)

or Sham-CAD as a control procedure. Three weeks later, a temperature sensor (TR3000 XM-FM; Mini-Mitter, Sun River, OR, USA) was implanted into the peritoneal cavity, and after recovery from this surgery, the rats were subjected to two experimental trials: resting under thermoneutral conditions (26 ◦ C) or heat exposure (36 ◦ C). The surgical procedures were performed under ketaminexylazine anesthesia (116 and 6 mg/kg body mass, respectively, i.p.). Immediately after the surgeries, the rats received an intramuscular prophylactic dose of antibiotics (pentabiotic, 48,000 IU/kg body mass) and a subcutaneous injection of analgesic medication (flunixin meglumine, 1.1 mg/kg body mass). Caudal artery denervation. The caudal artery was exposed by two 8 cm ventral incisions starting from the base of the tail: the first incision was made in the skin, followed by another in the subcutaneous tissue. After being visualized, the caudal artery was gently dissected from its surrounding tissue; care was taken to avoid sectioning the arteriovenous anastomosis. Then, the caudal artery was painted with a topical 10% phenol solution (diluted in glycerol), and the tail skin was sutured with absorbable thread (4.0). During the Sham-CAD surgery, the 8 cm ventral incision in the tail skin surface was made, and the skin surface was sutured without compromising any vascular innervation. Implantation of temperature sensors. A 2 cm ventral incision was made in the linea alba of the abdominal muscle, allowing for the insertion of a temperature sensor in the peritoneal cavity [8]. Following the insertion, the abdominal muscle and skin were sutured in layers. All animals were allowed to recover for 4 days before being subjected to the experiments. On the day of the experiments (26–28 days after CAD or ShamCAD surgery), each rat was weighed, a thermocouple was fixed to its tail surface, and the rat was placed inside an acrylic chamber (49.5 cm long × 14 cm wide × 13.5 cm high). An electrical fan positioned at one end of the chamber generated an air flow rate of 2.0–2.5 m/min. To heat the environment inside the chamber, an electrical heater (Britânia model AB 1100, Curitiba, PR, Brazil) was positioned at the same level 20–30 cm distant from the fan and turned on at 1200 W. During resting at 26 ◦ C, Tcore and Tskin were recorded for 60 min after their levels had stabilized; during heat exposure, the thermoregulatory parameters were recorded immediately after the animals were placed inside the environmental chamber. The intraperitoneal temperature was measured by telemetry and considered to be the Tcore index. Tskin was measured on the lateral surface ∼1 cm from the base of the tail [24] using a thermocouple. Tcore values were recorded every 10 s, whereas Tskin and Ta inside the chamber were recorded every min throughout the experiments. The heat loss index (HLI) was used to determine if the ambient temperatures studied corresponded to neutral or supraneutral conditions in our experimental setup [11] and calculated as [Tskin − Ta ]/[Tcore − Ta ]. To analyze the variations in both Tcore and Tskin , we calculated their coefficients of variation as [average standard deviation/pre-experimental values] (variability index 1) or their average standard deviation throughout experiments (variability index 2). Glyoxylic acid fluorescence histochemical method. At the end of the experiments (30 days after the CAD or Sham-CAD surgery), the rats were anesthetized with ketamine-xylazine (116 and 6 mg/kg body mass, respectively, i.p.), and 2 cm-long samples of the caudal artery were removed by ventral incisions at the base, middle and tip of the tail. The samples were analyzed using two different protocols: half of them were stored in −80 ◦ C freezer, and the other half were immediately analyzed. The first protocol was conducted to determine the location of sympathetic innervations in the caudal artery, and the second protocol was conducted to determine the density of the sympathetic innervations in the caudal

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artery. The samples stored in the freezer were cut into 10 ␮m rings using a cryostat microtome at −30 ◦ C. The rings were mounted on slides and immersed 3 times for 30 s in 2% glyoxylic acid solution (diluted in 0.1 M phosphate buffer adjusted to pH 7.4 with 10 M NaOH). The slices were dried for 20 min in a cold air stream. After being covered with mineral oil, the slides were heated at 60 ◦ C for 30 min and then coverslipped. The samples that were analyzed immediately were placed in fresh 9% saline, opened longitudinally and incubated in the same glyoxylic acid solution for 45 min. Each artery was then removed from this solution and stretched as a whole mount on a glass slide. The slices were dried, covered with mineral oil, placed in an oven, and processed according to the same procedures described above. Caudal arteries were visualized using a microscope equipped with an HB0 100 W mercury light source (Microscope Axioplan-Zeiss, Carl Zeiss GmbH, Oberkochen, Germany). The data are expressed as the means ± S.E.M. The differences in thermoregulatory adjustments were compared between groups and across time points by two-way analyses of variance, with repeated measures applied only for the time factor. When appropriate, the post hoc Tukey’s test was used for multiple comparisons. Unpaired Student’s t-tests were used to compare the body mass, Ta , and variability indexes for Tcore and Tskin between groups. The significance level was set at P < 0.05.

3. Results The effectiveness of caudal artery denervation was confirmed by the marked vasodilation of the caudal artery immediately after the topical application of phenol (Fig. 1A and B: representative images). This effectiveness was also confirmed 30 days after the surgical procedure by the absence of endogenous catecholamine-mediated fluorescence in both transverse (Fig. 1D) and longitudinal sections (Fig. 1F). In contrast, the Sham-CAD rats had high density of sympathetic innervation in the adventitial–medial junction (Fig. 1C) and in the longitudinal sections of their caudal arteries (Fig. 1E). It is important to emphasize that only the vascular portion treated with phenol was denervated; thus, the middle and tip of the caudal artery were not affected by the surgical procedure (data not shown). On the day of the experiments, the body mass was not different between groups (CAD 326 ± 7 g vs. Sham-CAD 310 ± 10 g; P = 0.218), indicating that chronic sympathectomy did not change the growth, hydration status or feeding pattern of the animals. The average Ta values were 25.6 ± 0.1 ◦ C and 35.5 ± 0.1 ◦ C for the resting and heat exposure experiments, respectively; no differences were observed in the Ta between groups during the same experimental trial. The average HLI values during resting and at the 60th min of heat exposure were 0.23 ± 0.03 and 0.61 ± 0.04, respectively. The HLI data indicate that the environmental conditions were within the thermoneutral zone during resting and were supraneutral during heat exposure [11]. The chronic absence of tail artery innervation did not change either Tcore (P = 0.997) or Tskin (P = 0.862) during rest under thermoneutral conditions (Fig. 2A). However, although the variability of Tcore was not different between groups, caudal artery denervation decreased the variability of Tskin (variability index 1: 0.020 ± 0.005 ◦ C vs. 0.031 ± 0.005 ◦ C; P = 0.024; variability index 2: 0.583 ± 0.043 ◦ C vs. 0.853 ± 0.134 ◦ C; P = 0.042; Fig. 2B). In the subsequent experiments, different animals from both groups were exposed to a Ta of 36 ◦ C for 60 min. In response to heat exposure, Tskin and Tcore increased by ≈6.0 and ≈1.6 ◦ C, respectively, in both Sham-CAD and CAD animals (Fig. 2C). No differences were observed in the Tcore values of both groups during heat

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exposure (P = 0.479). In contrast, although the peak values of Tskin were not affected by the chronic sympathectomy (CAD 38.1 ± 0.2 ◦ C vs. Sham-CAD 38.3 ± 0.4 ◦ C; P = 0.532), CAD animals showed a delayed increase in Tskin ; the time until the stabilization of Tskin was threefold longer in CAD rats than Sham-CAD rats (15.3 ± 2.5 min vs. 4.9 ± 0.6 min; P = 0.001; Fig. 2D). Therefore, Tskin was lower in CAD rats than ShamCAD rats from the 2nd until the 10th min of heat exposure (Fig. 2C).

4. Discussion The present findings show that chronic sympathectomy of the caudal artery induced a slower increase in Tskin when the animals were passively heated and a decrease in Tskin variability when the animals were allowed to rest under thermoneutral conditions. Interestingly, despite the delayed increase in Tskin in denervated rats during heat exposure, no differences were observed between groups with respect to the maximum values achieved (≈38.0–38.5 ◦ C). Moreover, heat exposure-induced hyperthermia was not altered by caudal artery denervation. Although we did not observe differences in the average values of Tcore and Tskin , CAD rats showed a reduction in Tskin variability under neutral conditions (Fig. 2B). This result suggests that although chronic adaptations may have restored the average Tskin of CAD rats to values similar to those of ShamCAD rats, the fine-tuned control of heat exchange between the skin and the environment was irreversibly impaired by sympathectomy of the caudal artery. Under neutral conditions, the diameters of the arterioles that control cutaneous blood flow exhibit substantial fluctuations, changing between states of vasoconstriction and vasodilation; these changes in the cutaneous vasomotor tonus tightly control Tcore and drive the fluctuations in Tskin [11]. The observation that fluctuations in Tskin have a high frequency indicates that this fine-tuned control is dependent on neural rather than on humoral mechanisms. Therefore, it is possible that this reduction in Tskin variability is related to the loss of activity from sympathetic preganglionic neurons controlling the tail vasculature. Supporting our hypothesis, Kalincik et al. [4] attributed the decrease in tail blood flow variability induced by spinal cord lesions to the absence of neural control over cutaneous vessels. A previous investigation of the effects of spinal cord injuries on the responses to cold exposure showed a slower reduction in blood flow in injured animals [4]. Further evidence regarding the importance of sympathetic control on cutaneous heat loss came from investigations showing that cutaneous vasodilatation in a rat’s tail primarily occurs due to the withdrawal of sympathetic vasoconstrictor activity over the skin vessels [7]. Based on these findings, our CAD rats should have presented higher cutaneous heat loss when exposed to warm environments, which was not observed in the current study. In fact, the opposite response was observed: the increase in Tskin of CAD rats was markedly slower than that observed for control rats (Fig. 2C). This unexpected finding may be a consequence of the adaptations that usually develops with time in denervated rats, preventing the development of a severe, life threatening hypothermia. To prevent abrupt reductions in Tcore , thermoeffector that promote heat production can be engaged (i.e., increase in spontaneous motor activity, shivering or brown adipose tissue thermogenesis [4]). In parallel, functional and morphological vascular adaptations aimed at decreasing heat loss to the environment can also be engaged, inducing greater vascular sensitivity to circulating and local vasoconstrictor agents (catecholamines, angiotensin II and endothelin-1 [19,21]). Considering that the increase in tail heat loss in CAD rats must be

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Fig. 1. Confirmation of the effectiveness of caudal artery denervation. During surgery, caudal artery denervation was confirmed by marked vasodilation induced by the topical application of phenol. Panels A and B show images of the caudal artery from a rat subjected to the denervation procedure before and after phenol application, respectively. Thirty days after the surgical procedure, the caudal artery denervation effectiveness was confirmed by the absence of endogenous catecholamine-mediated fluorescence at the adventitial–medial junction in transverse sections (Panel C: representative Sham-CAD rat; Panel D: representative CAD rat) and longitudinal sections (Panel E: Sham-CAD rat; Panel F: CAD rat). Red arrows indicate sympathetic innervation, and blue arrows indicate the autofluorescence of internal elastic lamina. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

mediated by local and humoral mechanisms, the adaptive changes favoring cutaneous vasoconstriction may have attenuated the effects of local and humoral vasodilator agents, contributing to the delayed increase in Tskin . Although the CAD rats exhibited a delayed increase in Tskin , the maximum values achieved during heat exposure were not different between groups (Fig. 2C). We suggest that after the initial minutes of heat exposure, when cutaneous vasodilation is primarily a consequence of the withdrawal of the sympathetic outflow over skin vessels, local and humoral mechanisms also play important roles in the modulation of cutaneous heat loss.

Supporting this hypothesis, it has already been demonstrated that thermal stimuli (heat locally applied to the skin and/or increases in Tcore ) increase cutaneous blood flow [10]. Even when isolated from each other, both thermal stimuli are involved in the reflex inhibition of sympathetic activity and in the endothelial production of nitric oxide, which spreads to vascular smooth muscle, producing vasodilation [13,18]. Therefore, we suggest that the vasodilator stimuli triggered by heat exposure may have overcome the vascular adaptations associated with denervation, increasing Tskin of the CAD rats to values similar to those recorded in the control animals.

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Fig. 2. Thermoregulatory adjustments in neutral and warm environments. Temporal profiles of Tcore and Tskin of Sham-CAD and CAD rats during rest at a Ta of 26 ◦ C (Panel A) and during passive heating at a Ta of 36 ◦ C (Panel C). Panel B shows the variability of Tcore and Tskin under thermoneutral conditions; whereas the panel D shows the time elapsed until Tskin plateaued and the peak value of Tskin in warm conditions. *P < 0.05 compared with Sham-CAD rats.

Interestingly, Tcore was not affected by chronic sympathectomy of the caudal artery, despite the slower increase in Tskin during the initial minutes of heat exposure. As previously reported, in addition to cutaneous vasodilation, the spreading of saliva (which may lead to saliva evaporation) is an effective mechanism through which rats dissipate heat to the environment, allowing for the maintenance of Tcore during passive heating [16]. Therefore, we hypothesized that CAD rats could maintain their Tcore during heat exposure by enhancing saliva production and/or salivary grooming. Stricker and Hainsworth [16] demonstrated that after partial tail amputation, rats increase the level of evaporative cooling during heat stress by salivary grooming, a finding that supports our hypothesis. It is important to emphasize that our animals were subjected to 60 min of passive heating; more prolonged heat exposures (i.e., 3–4 h) that

are severe enough to reduce the plasma volume may limit saliva production [25], leaving the CAD rats more vulnerable to heat illnesses. Questions can be raised regarding whether the sensory nerves in the ventral caudal artery were also lesioned, since phenol is a nonselective neurodegenerative drug [22]. However, both anatomical and physiological evidence indicates that sensory nerves do not contribute to the regulation of vascular tonus in the caudal artery. The sensory fibers present in the tail are more likely to arise from the skin than from the caudal artery [14]. Supporting this finding, Li and Duckles [5] reported that the activation of sensory nerves (through the local release of CGRP) did not affect the vascular tone of the caudal artery. Moreover, different from what takes place in the other tail vessels, the caudal artery sensitivity to noradrenaline

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did not change when local temperature was altered [10]. This finding suggests that the perivascular sensory nerves of the caudal artery (if existent) are not stimulated by temperature. Accordingly, Slovut et al. [15] demonstrated that the vascular adaptations induced by a depletion of catecholamines in the peripheral sympathetic nerve endings were similar to those induced by a topical application of phenol to the ventral caudal artery, indicating that the perivascular nerves of this vessel are predominantly sympathetic. In conclusion, chronic sympathectomy of the caudal artery delayed the increase in Tskin in response to heat exposure, suggesting that both the lack of sympathetic inputs and the vascular adaptations induced by denervation can transiently impair cutaneous heat loss during passive heating. In addition, CAD rats showed a decreased variability of Tskin , indicating that sympathetic outflow to the tail provides fine-tuned control over cutaneous heat loss. Taken together, these results demonstrate that the intact sympathetic innervation of cutaneous vessels is essential for the precise and fast regulation of tail heat loss. Acknowledgments The authors are indebted to the CNPq (Brazil), FAPEMIG and Pró-Reitoria de Pesquisa da Universidade Federal de Minas Gerais for financial support. M.R.M.L., W.P., I.A.T.F. and C.G.F. were recipients of fellowships from the CAPES (Brazil). We thank Dr. Elisabeth Ribeiro da Silva from the Department of Morphology for kindly providing the microscope and reagents used for the histochemical analysis. We also thank Carlos Henrique da Silva for technical support during the histochemical analysis.

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