The impact of temperature on vascular function in connection with vascular laser treatment

Eighteen Lewis rats (9 female, 9 male) aged 12 to 16 weeks, weighing 270 ± 55 g were deeply anesthetized using isoflurane (isoflutek 1000 mg/g, Karizoo, Barcelona, Spain) and after subsequent decapitation, second and/or third-order mesenteric arteries, each approximately2 mm in length, were dissected with dissection tools (student dissection kit 504167, world precision instruments, WPI-Europe, Friedberg, Germany) and isolated from the rat mesenteric vascular bed (Fig. 1). All procedures were conducted in accordance with the guidelines of the National Institute of Health (NIH) for the care and use of laboratory animals and institutional protocols.

The presence and morphological integrity of endothelial cells, smooth muscle cells, and vascular nerves were confirmed by fluorescence immunohistochemistry. One rat was anesthetized and infused with Lectin conjugated to AF594 (DL-1177, Vectorlabs, Vector Laboratories, Newark, United States)) to stain for endothelial cells and then fixed with PFA to prevent a collapse of the blood vessels. Vessel segments were fixed in Zamboni Fixative (263-01991, Bio-connect,FUJIFILM Wako, Neuss, Germany) for 24 h at 4°C and then washed three times in dPBS (PBS-1A, Capricorn Scientific, Capricorn Scientific, Germany). Blocking of non-specific antigen binding was performed with 4% normal goat serum (X090710-8, Agilent, Agilent technologies, Amstelveen, The Netherlands) and 1% Triton X-100 (9036-19-5, BOOM, Merck, The Netherlands) in PBS for 2 h at room temperature. Then, vessels were incubated with rabbit anti-rat protein gene product 9.5 (PGP9.5) 1:500 (ab108986, Abcam, Cambridge, United Kingdom), Neuronal peptide Y (NPY-EPR21877, ab221145, abcam, Cambridge, United Kingdom,), or calcitonin gene-related peptide (CGRP-4901, ab81887, abcam, Cambridge, United Kingdom) overnight at 4°C. Primary antibodies were diluted in a 1:1 mixture of PBS and blocking solution. Vessels were washed thrice with PBS and concordantly incubated with goat anti-rabbit cy3 (111-165-144, Brunschwig, Jackson immune Research, Cambridgeshire, United Kingdom), at 1:800 dilution in PBS for 2 h at RT. Vessels were rinsed in milliQ (CDUFBI001 Q-Pod, Biopak, Merck, The Netherlands) and mounted on a glass slide in DAPI vectashield (H-1200-10, Vectashield, Vector Laboratories, Newark, United States). A 3D set of images were obtained with a Leica SPM-8X Confocal Microscope (DLS lightsheet, Leica, Leica Microsystems BV, Amsterdam, The Netherlands) equipped with a 40x/1.3 OIL objective.

The isolated arteries were positioned in a wire-myograph setup (620M, Danish Myo Technology, DMT, Aarhus, Denmark) to measure the contractile force generated by the artery under different conditions. Rat mesenteric first and second-order arteries were used with a mean diameter of 360 ± 60 μm and a mean vessel length of 1.9 ± 0.1 mm. Vessels were exposed to various temperatures and subsequently further stimulated using either agonists or transversal electrical field stimulation (EFS) by electrodes next to the blood vessel. The wire-myograph was also equipped with an in-house assembled device using K-type thermocouples (621-2158, RS, RS Components BV, Haarlem, The Netherlands) that measured temperature fluctuations in each vessel chamber, calibrated for a temperature range of 20°C to 70°C.

At the start of the experiment, vessels were equilibrated to 37°C, and the vessel was pre-stretched in calcium-free MOPS buffer (for buffer composition see supplemental Table 1). Calcium-free conditions were used here to prevent smooth muscle contraction. This procedure was used to obtain the optimal level of pre-stretch for each vessel, which is at 90% of the diameter at 100 mmHg. At this level of pre-stretch vascular responses are maximal. [17, 18] The result of this procedure is a passive force of the vessel around 3 to 4 mN. Then, vessels were acclimatized for ten minutes in 37°C physiological salt solution (PSS) (see components in supplemental Table 1) and the chamber was aerated with a gas mixture of 95% air and 5% CO₂ (UN1956, 1470110, Lindegas, The Netherlands) to maintain metabolic activity of the cells. Vessels were then exposed shortly to 125 mmol / L potassium solution (KPSS, see supplemental Table 1) to test for viability, rinsed thrice with PSS, and rested for five minutes. Vessels demonstrating a contractile force of less than 10 mN to the non-receptor-mediated stimulation of smooth muscle cells by KPSS were considered to be damaged and excluded from the experiment. The vessels were then contracted with 1 x 10^-5 μmol U46619 (L-thromboxane A₂ agonist, D8174, Sigma Aldrich, Amsterdam, The Netherlands). The contracted state upon exposure to U46619 was then used to assess vascular relaxation through the addition of 1 x 10^-5 mol/L methacholine (A2251, Sigma Aldrich, Amsterdam, The Netherlands), which is an endothelium-dependent vasorelaxing agent (non-selective muscarinic receptor agonist). The chamber was washed thrice with PSS and the vessels were rested for five minutes. Lastly, electrical field stimulation was used to stimulate nerve endings to initiate smooth muscle cell-mediated contraction. EFS potentially stimulates both nerves and smooth muscle cells at the same time, hampering the possibility of assessing the involvement of nerves in the response. Therefore, the frequency of EFS that directly induces smooth muscle contraction was determined by using a nerve blocker (3 μM TTX, HB1035, HelloBio, Dunshaughlin, Ireland ). Blocking nerve function while applying EFS showed that smooth muscle contraction appears at 32 Hz and higher frequencies (Supplemental Fig. 2). Therefore, our experiments were performed in the nerve-specific range of 0.5 Hz – 16 Hz. In more detail, the frequency consisted of a range of 0.5, 1, 2, 4, 8, and 16 Hz, each with a pulse duration of 0.4 ms and 40 mA, and pulse lengths of 10 sec with 1-sec breaks between incrementing frequencies (0.5Hz to 1 Hz, 1Hz to 2Hz, etc). Vessels were stimulated by three sets of EFS with a 120-second rest in between. Repeating the EFS three times allowed the blood vessel to reach the maximal contraction. Vessels were then rested for 10 minutes and subsequently exposed to heated PSS in the range of 42°C – 62°C, or kept at 37°C (control). The heated PSS was administered into the chamber by a pre-warmed plastic syringe. After 30 seconds, the PSS was replaced with PSS at 37°C, and vessels were rested for 5 minutes. Then, the protocol was repeated (KPSS, U46619, methacholine, EFS). Maximum values of individual vessels were compared before and after hyperthermia.

Maximal contraction force was retrieved using the max-min function in Chart 5 (V5.5.6, ADInstruments, Oxford, United Kingdom). Maximal force (in mN) after heating was divided by the maximal force before heating, yielding a ratio for each response that was used to assess hyperthermia-induced functional damage. Averaging these ratios from individual vessels resulted in a mean ± SEM of relative response R as shown in Fig. 4. A non-linear four-parameter fit of relative response R(T) forced through zero was applied to the individual data points with R_max, ET50 and γ as fitting parameters to find the temperature at which 50% of the vessel functionality was lost (Effective Temperature, ET50): \(R(T)=\frac{R_{max}}{\left(1+{10}^{\left( LD50-T\right)\bullet \gamma}\right)}\) where R_max is the maximum response, and γ the decrease in relative response (contraction or vasorelaxation) per °C increase in temperature. To compare the ET50 for the four stimuli we performed a Least Squares regression analysis with multiple comparison testing and Bonferroni correction, α = 0.05 . We compared all three cell types by testing the ET50 values of KPSS vs Methacholine, KPSS vs EFS, and EFS vs Methacholine. Here, the cut-off value for statistical significance was adapted for three comparisons at 0.05 / 3 = 0.017. ET50 values are shown with their symmetric 95% confidence interval (ET50 ± CI95). All statistical analyses and data visualization were performed using GraphPad Prism Software v.8.3 (Prism, Graphpad, Graphpad Software, Boston, Massachusetts, United States).

Immunohistochemistry confirmed the presence of endothelial cells, smooth muscle cells, and vascular nerves within the rat mesenteric artery, showing the morphology of the vessel wall and its integrity after dissection. As depicted in Fig. 2, the inner lining of the blood vessel is formed by endothelial cells (marked in red, lectin), followed by a layer of smooth muscle cells (marked in blue, α-SMA), and a plexus of vascular nerves (marked in green, PGP-9.5) (supplementary video 1). A closer examination of the vascular nerve plexus revealed a double-layered structure. The first layer consisted of thinner, sympathetic, nerve fibers positive for neuropeptide Y (NPY), that were nestled between the smooth muscle cells. The outer layer of the nerve plexus contained thicker and less dense (sensory) nerve fibers positive for calcitonin gene-related peptide (CGRP) (Supplementary Fig. 1).

Fig. 3 shows the force produced by the vessel upon stimulation with KPSS, U46619, Methacholine, and EFS in time and as a function of temperature. Each blood vessel was exposed only once to a specific temperature. The first frame in Fig. 3 shows that KPSS stimulation resulted in the strongest and most consistent contraction among all stimuli. The KPSS was then washed out thrice, resulting in three small increases in force due to the flow of liquid within the measurement chamber. The second frame in Fig. 3 shows the contraction as a response to U46619. This pre-contracted state was then used to assess the endothelial-mediated vasorelaxing response to methacholine resulting in a decrease in force (mN) measured. Finally, the last frame shows the effect of nerve stimulation and consecutive vasocontraction during EFS. The EFS-frame of the vessel exposed to 55°C shows that there is incomplete relaxation after EFS stimulation. For all stimuli the response was decreased after ~55°C and abolished at ~60°C.

The non-receptor-mediated contractile force induced by KPSS was maintained for the first part of the temperature range but noticeably decreased after exposure to 55°C (Fig. 4). The temperature at which the KPSS response was reduced by 50% (ET50) was calculated at 56.9°C (56.2-57.7°C), with a goodness of fit (R²) of 67% (Fig. 4, supplemental Table 2). Receptor-mediated stimulation of smooth muscle cells by U46619 led to a decline in contractile force after exposure to temperatures around 50°C. The ET50 for U46619-induced contraction was calculated to be 51.5°C (47.0-55.8°C), with an R² of 34%. In some instances, the loss of contraction occurred even before heating was applied or upon exposure to 37°C. Consequently, this inconsistency in response patterns affected the 95% confidence interval for the ET50 of the U46619 reaction, which ranged from 47.0°C to 55.8°C.

The maximal response to nerve-regulated smooth muscle cell contraction by EFS was consistently observed to be at its maximum at 16 Hz and exhibited a decline in response after exposure to 56°C – 60°C. In comparison to KPSS, EFS-induced contractions were lower and did not exceed a response of 6 mN. Fitting the data to obtain the ET50 of EFS generated a value of 55.9°C (55.1-55.6°C), and R² of 50%. Additionally, we observed a decrease in the variability of response to EFS at around 58°C. The fit for EFS displays a slightly steeper slope γ (-0.58 compared to -0.23 per °C) and a 1°C lower ET50 compared to KPSS-challenged vessels (supplemental Table 2, Fig. 4). Yet, there was no statistically significant difference amongst ET50 of KPSS and EFS (p=0.06), nor between the ET50 of methacholine and EFS (p=0.37).

Assessment of the maximal vasorelaxation induced by methacholine was feasible only when U46619 pre-contraction was present. In the majority of the dataset, methacholine resulted in full vasorelaxation at temperatures where U46619 already showed a declined contraction (as observed in Fig. 3 at 50°C and 55°C). As methacholine is endothelium-dependent, this finding suggests that endothelial cells can retain their functionality even after exposure to temperatures around 55°C. The slope γ of methacholine-induced endothelial-mediated relaxation was -0.09 per °C, showing a much slower decrease in functionality compared to KPSS-stimulated smooth muscle cells. Methacholine demonstrated an ET50 value of 56.7°C (55.5-58.1°C), with an R² of 48%. There was no statistical significant difference between the ET50 of KPSS and methacholine (p=0.82), nor between methacholine and EFS (p=0.37).