Lumbar sympathectomy reduces vascular permeability,possibly through decreased adenosine receptor A2a expression in the hind plantar skin of rats

Abstract

BACKGROUND:

The effect of lumbar sympathectomy for the treatment of lower limb ischemia remains a matter of controversy.

METHODS:

Sprague-Dawley rats were subjected to lumbar sympathectomy, after which Evans blue dye was injected into the hind plantar skin. Extravasation of dye was measured and compared with rats undergoing sham operation. Hind plantar skin was processed for HE staining, immunohistochemistry, and Western blot.

RESULTS:

In sympathectomized rats, blue stained areas in hind plantar skin and concentrations of Evans blue were significantly less than that of sham sympathectomy (control) rats, both 2 weeks and 3 months after surgery. Expression of prostaglandin E2, bradykinin, bradykinin B2 receptor, and adenosine triphosphate were not significantly different between the sympathectomized and control groups. Adenosine receptor A2a expression was significantly reduced in the sympathectomized group both 2 weeks and 3 months after surgery.

CONCLUSION:

Vascular permeability in the hind plantar skin of rats decreases following lumbar sympathectomy, possibly via reduced expression of adenosine receptor A2a.

Keywords

Lumbar sympathectomy vascular permeability Sprague-Dawley rat Evans blue adenosine receptor A2a

1 Introduction

Previous studies have shown that blood vessel permeability is inversely related to sympathetic nerve activity. Repeated, non-habituating stress significantly increased sympathetic nerve activity, and inhibited bradykinin-induced plasma extravasation in rats [1]. Green KL [2] suggested that sympathetic catecholamine neurotransmitters inhibit edema in mouse hind paw induced by carrageenan and 5-hydroxytryptamine.

Some question remains as to whether vascular permeability is altered following failure of sympathetic nerve function. Leis S [3] showed that BK-induced protein extravasation in forearm skin increased after preganglionic endoscopic transthoracic sympathetic clipping at the T2 or T3 level in humans. However, Engel D [4] found synovial membrane permeability decreased on the operated side in unilaterally sympathectomized cats.

In this study, we evaluated capillary permeability changes in the hind plantar skin of rats following lumbar sympathectomy. We used a Evans blue fluorescence assay to explore the effects of sympathectomy on vascular permeability. We measured expression of vascular permeability regulators, including adenosine triphosphate (ATP), adenosine receptor A2a(A2AR) [5], bradykinin receptor B2 (BDKRB2) [3], bradykinin (BK), and prostaglandin E2(PGE2) [6, 7].

2 Materials and methods

2.1 The operation of lumbar sympathectomy

Thirty-two Sprague-Dawley rats, weighing 250–300 g, were purchased from the experimental animal center of Southern Medical University, Guangzhou, China. Rats were randomly assigned to lumbar sympathectomy and sham sympathectomy (control) groups. Rats were allowed to adapt at least 1 week prior to experiment. The animals were maintained at 22–24°C with a 12 h light/dark cycle and ad libitum access to standard laboratory food and water. The experiments were conducted according to institutional guidelines and were approved by the animal care committee.

Rats in the sympathectomy groups underwent bilateral lumbar L2-4 sympathetic trunk resection as follows: animals were anesthetized by intraperitoneal injection of 0.6% pentobarbital sodium (40 mg/kg, i.p.; Anthony Products, Arcadia, CA, USA). After shaving and disinfection, a midline incision was made from the xiphoid to the pubic symphysis. Lumbar sympathetic trunks were located behind the abdominal aorta and vena cava and were dissociated under a surgical microscope (Leica M300, Germany) from the level of the left renal artery (L2 sympathetic ganglia) to the level of the descending aorta bifurcation (L5 sympathetic ganglia) [8]. Bilateral L2-L4 sympathetic trunks were cut and sent for frozen sections for hematoxylin and eosin (HE) staining and for detection of the sympathetic marker tyrosine hydroxylase (TH) expression. After complete hemostasis, the incision was closed with 5–0 suture. Animals were then transferred to a clean cage. Continuously throughout the process, the temperature of left hind plantar skin was recorded with a point monitoring thermometer (NeXus-10 MKII, Stens Biofeedback – Stens Corporation, San Rafael, CA, USA). Sham operated animals (control group) underwent dissociation, but not resection of the sympathetic trunks.

2.2 Evans blue assay

Evans blue fluorescence is a method of measuring vascular permeability in animal tissues with high sensitivity and accuracy [9]. Three rats in each group were anesthetized at 2 weeks and 3 months following surgery. Evans blue (20 mg/kg, 4 ml/kg) [10] was injected within 1 minute into the tail vein. The hind plantar skin was photographed 40 minutes following injection. Full-thickness hind plantar skin specimens (0.8 cm) were cut into pieces and placed in 1 ml formamide at 37°C for 72 hours. Specimens were then centrifuged at 3000 r/min for 15 min at 4°C. The absorbance of the supernatant was then measured at 610 nm in a Varian UV-VIS spectrophotometer. Concentration of Evans blue were calculated according to standard curves.

2.3 Plantar skin resection

The remaining five rats in each group were anesthetized 2 weeks or 3 months after surgery. Two round full-thickness skin (0.8 cm diameter) from both sides of the hind plantar skin resected cut for HE stain, immunohistochemistry and Western blot.

2.4 Histology evaluation of lumbar sympathetic trunks and plantar skin

For histological analyses, harvested samples of hind plantar skin were fixed in 4% formaldehyde in PBS at 4°C, dehydrated in a graded series of ethanol, then embedded in paraffin for routine HE stain [11]. Fresh frozen sections of lumbar sympathetic trunks were used for HE staining. HE stained sections were observed with a light microscope (Olympus BX51, Japan).

2.5 Immunohistochemistry of lumbar sympathetic trunk and plantar skin

Fresh frozen sections of lumbar sympathetic trunks in rats were sectioned (5 μm) and fixed with ice cold acetone for 10 minutes. The sections were immersed in 3% hydrogen peroxide in methanol to block endogenous peroxidase activity, and were then were immersed in EDTA buffer (pH 6.0) for 20 min at 95°C. After the sections were blocked by 5% fetal bovine serum, sections were stained with anti-goat polyclonal TH antibody (1:800 dilution; Abcam, Cambridge, UK). The primary antibodies were incubated for 1 hour at 25°C.

Paraffin sections (5 μm) of sacrococcygeal skin were de-paraffinized, washed three times in PBS for 5 min, then blocked with 5% bovine serum for 30 min. The slides were subsequently incubated with primary antibodies against pan-ATP (H-39) (1:100 dilution; Santa Cruz, CA, USA), BK (1:100 dilution; Santa Cruz, CA, USA), PGE2 (1:200 dilution; Abcam, Cambridge, UK), BDKRB2 (1:200 dilution; Novus, CO, USA), and A2AR (1:200 dilution; Novus, CO, USA) at 4°C overnight.

After rinsing three times with PBS, slides bearing lumbar sympathetic trunks and plantar skin were incubated with secondary antibodies at 37°C for 30 min. The secondary antibodies against TH, PGE2, BDKRB2 and A2AR were horseradish peroxidase-labeled antibodies, further developed with 3,3’-diaminobenzidine tetrahydrochloride (DAB) solution. The secondary antibodies against pan-ATP and BK were Alexa Fluor 488-conjugated antibodies. Antibody against PGE2 was not available for Western blot, so we measured integrated optical densities (IODs) of stained areas using Image Pro Plus 6.0 software [12].

2.6 Western blot of plantar skin proteins

The hind plantar skin tissue lysate was homogenized in RIPA buffer (50 mM Tris HCl, pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS,2 mM EDTA, protease inhibitor cocktail, phosphatase inhibitor cocktail, and 1 mM DTT). Protein concentration was determined using the BSA method. Skin lysates were denatured at 95°C, for 5 min in sample buffer. Forty μg of total protein was resolved on 12% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% fat-free dry milk in Tris-buffered saline containing 0.1% (v/v) Tween 20 (TBS-T) for 1 h at room temperature. After blocking, membranes were incubated with primary antibodies against pan-ATP (H-39) (1:100 dilution; Santa Cruz, CA, USA), BK (1:3000 dilution) and BDKRB2 (1:1000 dilution; Novus, Co, USA) and A2AR (1:1000 dilution; Novus, Co, USA) overnight at 4°C. After incubation, membranes were washed and incubated for 1 h at room temperature, with anti-goat antibody (1:20000 dilution) or anti-Rabbit antibody (1:20000 dilution). The membranes were exposed to ECF reagent, followed by scanning on the VersaDoc (Bio-Rad Laboratories, Portugal). For normalization, the membranes were re-probed with an anti-GAPDH antibody (1:10000 dilution). Generated signals were analyzed using the Image-Quant TL software.

2.7 Statistical analysis

Data are expressed as mean±standard deviation (SD). Statistical comparisons were performed using ANOVA (t-test). All statistical computations were performed using SPSS software (ver. 21.0; SPSS, Chicago, IL). Differences with a value of P < 0.05 were considered statistically significant.

3 Results

3.1 Confirmation of lumbar sympathetic trunk resection

One rat in the lumbar sympathectomy group and another in the control group died from hemorrhage during surgery. Lumbar sympathetic trunks were pale in color and were located anterior to the spine, medial to the genitofemoral nerves, and posterior to the abdominal aorta and vena cava (Fig. S1), consistent with a prior study [13]. The skin temperature of the left foot increased intraoperatively above 0.8°C 30 minutes following lumbar sympathetic trunks resection. This temperature change was used as a marker of successful sympathetic trunks resection [14 –16] (Fig. S2). HE staining results of lumbar bilateral sympathetic trunks are shown in Fig. S3. The presence of TH staining suggests the presence of sympathetic neuronal tissue (Fig. S4).

3.2 Evans blue assays

There was a smaller blue-stained area in the hind plantar skin of the sympathectomized group compared with that of the control group (Fig. 1A). The concentrations of Evans blue in sympathectomized rats were lower at both 2 weeks (P = 0.004) and at 3 months (P = 0.041) compared with control rats (Fig. 1B). The concentrations of Evans blue within each group at 2 weeks and 3 months were not significantly different (Table 1).

Fig.1

The level of vascular permeability could be assessed by simple visualization(A) or by quantitative measurement of the Evans blue dye(B).

Table 1

The quantitative measurement of the Evans blue dye in the skin of hind feet in rats

	2 weeks	3 months
Control	3.05e-3±1.01e-3	2.89e-3±9.31e-4
sympathectomized	1.33e-3±5.29e-4	1.67e-3±8.64e-4
P Value	0.004	0.041

3.3 Immunohistochemistry of plantar skin

By immunofluorescence, ATP levels in the experimental group were not different from those of the control group (Fig. 2A-D). Scant expression of BK was observed in the hind plantar skin, especially in the sympathectomized group 2 weeks after surgery (Fig. 2E-H). BDKRB2, A2AR, and PGE2 were expressed in the endothelium of arteries and veins, capillaries, epidermal cells, and sweat gland epithelial cells (Fig. 3). We also measured PGE2 in plasma. The IODs of PGE2 were not significantly different between the sympathectomized and the control groups both at 2 weeks (P > 0.05) and at 3 months (P > 0.05) after surgery (Fig. 4, Table 2).

Fig.2

The results of the immunofluorescence detection of ATP and bradykinin. The bar corresponds to 50 μm.

Fig.3

The results of immunohistochemical staining of BDKRB2, A2AR and PGE2. The bar corresponds to 100 μm.

Fig.4

The IODs of PGE2 were not significantly different between the sympathectomized group and the control group 2 weeks and 3 months after modeling.

Table 2

The IODs of PGE2 in the skin of hind feet in rats

	2 weeks	3 months
Control	63865.68±15624.05	64411.30±56073.75
Sympathectomized	75688.95±43638.75	81213.36±98811.32
P Value	>0.05	>0.05

3.4 Western blotting of plantar skin proteins

Expression of BK, BDKRB2 and ATP (Fig. 5A-E) was not significantly different between the sympathectomized group and the control group both at 2 weeks (P > 0.05) and at 3 months (P > 0.05) after surgery (Fig. 5A-E). A2AR expression was lower in the experimental group both at 2 weeks (P = 0.044) and at 3 months (P = 0.026) after sympathectomy (Fig. 5A,F, Table 3).

Fig.5

The protein expressions of BK, ATP, BDKRB2 and A2AR were detected by western blot. ^#p < 0.05 The sympathectomized group compared with the control group.

Table 3

The semi quantitative value of western blot in the skin of hind feet in rats

Protein	2 weeks			3 months
	Control	Sympathectomized	P Value	Control	Sympathectomized	P Value
ATP	0.102±0.077	0.112±0.1045	>0.05	0.200±0.042	0.167±0.113	>0.05
A2AR	0.164±0.132	0.000±0.000	0.044	0.2446±0.081	0.114±0.059	0.026
BK	0.113±0.144	0.210±0.267	>0.05	0.353±0.0556	0.463±0.254	>0.05
BDKRB2	0.124±0.047	0.261±0.275	>0.05	0.075±0.011	0.175±0.192	>0.05

4 Discussion

Sympathetic nerves may participate in the processes of neurogenic inflammation [17] and subsequent arterial regeneration [18]. Sympathetic innervation is heterogeneously distributed among blood vessels [19]. Lumbar sympathectomy is a common intervention used to study the relationship between the sympathetic nervous system and the circulatory system. Following lumbar sympathectomy, peripheral blood vessels dilate in patients with critical limb ischemia [16]. Whether lumbar sympathectomy is useful for the treatment of lower limb ischemia remains controversial.

Vascular permeability can be assessed by simple visualization or by quantitative measurement of the Evans blue dye [20]. Endothelial permeability is regulated by beta-adrenoceptor agonists [21]. In our study, visualization and Evans blue absorbance in hind plantar skin significantly decreased following lumbar sympathectomy. This suggests that the vascular permeability of the hind plantar skin decreased following resection of the sympathetic trunk. This result is consistent with a previous report [4].

Expression of BK, bradykinin B2 receptors, ATP, and PGE2 was not significantly different at either at 2 weeks or 3 months post-sympathectomy between groups. This suggests that these molecules may not be involved in the observed decreased vascular permeability of the foot following sympathetic denervation.

In our study, A2AR expression decreased in the experimental group at both 2 weeks and 3 months following sympathectomy. 6-hydroxydopamine reduces norepinephrine (NE) concentration [22]. The simultaneous blockade of the A2AR and adenosine A3 receptor receptors significantly increases the release of noradrenaline [23]. Selective A2AR agonists increase permeability of the blood-brain barrier in murine models [24]. Therefore, we believe the decrease in A2AR may be a negative feedback signal for the decreases in norepinephrine, giving rise to reduced level of vascular permeability following sympathectomy.

Some investigators believe lumbar sympathectomy ameliorates lower limbs varicose disease, trophic ulcers [17 , 25–27] and ischemic diabetic foot disease [28, 29, 28, 29]. However, others believe the positive effects of lumbar sympathectomy on critical limb ischemia may be limited [30]. Whether lumbar sympathectomy is useful for the treatment of lower limb ischemia remains controversial. Our results suggest that vascular permeability in the plantar skin of rats decreases following lumbar sympathectomy. This effect may be mediated via reduced A2AR expression. Further study of this phenomenon is necessary to clarify the relationship between sympathectomy and critical limb ischemia.

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Acknowledgments

This work was supported by the National Natural Science Foundation of China, No. 81171812, 81272105 and 81671924.

The supplementary material is available in the electronic version of this article: .

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