Quantification of basal digital blood flow and after cold stimulus by laser doppler imaging in patients with systemic sclerosis

Abstracts

OBJETIVO: Determinar o comportamento dinâmico do fluxo sanguíneo da microcirculação digital, antes e após dois estímulos frios (EF) de diferentes intensidades, utilizando o método do Laser Doppler Imaging (LDI) em pacientes com esclerose sistêmica (ES) e controles saudáveis. MÉTODOS: Foram incluídos 14 pacientes com ES (51,2 ± 5,5 anos de idade) e 12 controles saudáveis (44,8 ± 9,9 anos). Foram realizados dois protocolos alternativos de EF (submersão das mãos em água a 10 ºC ou 15 ºC, durante 1 minuto). O fluxo médio das quatro polpas digitais da mão esquerda (FPD) foi mensurado com a utilização do LDI (Moor LDI-VR), em condições basais, nos períodos de 1, 4, 10, 25 e 40 minutos após EF. RESULTADOS: O fluxo basal foi significativamente menor em ambos os protocolos em pacientes com ES comparados a controles (312,9 ± 102,7 versus 465,4 ± 135,4 PU, P = 0,006, no protocolo a 15 ºC; 305,2 ± 121,0 versus 437,9 ± 119,8 PU, P = 0,01, no protocolo a 10 ºC). Nos controles houve declínio significativo do FPD após EF, em comparação aos valores basais apenas no tempo de um minuto após EF a 15 ºC (P = 0,001) e nos tempos de 1 e 25 minutos após EF a 10 ºC (P = 0,005; P = 0,001, respectivamente). Nos pacientes com ES, houve declínio significativo do FPD nos tempos de 1, 4 e 10 minutos após ambos EFs (P < 0,000; P = 0,002; P = 0,014, EF a 15 ºC; P < 0,000; P = 0,004; P = 0,001, EF a 10 ºC, respectivamente). CONCLUSÃO: LDI demonstrou baixa perfusão em polpa digital em condições basais e elevado declínio de perfusão com retardo na recuperação após EF na ES. A quantificação do fluxo sanguíneo pelo LDI pode ser útil para o seguimento longitudinal da doença e para a monitoração de intervenções terapêuticas na ES.

esclerose sistêmica; fenômeno de Raynaud; microcirculação; Laser Doppler imaging

ORIGINAL ARTICLE

^IPost-graduate Rheumatology student at UNIFESP

^IIProfessor of Rheumatology at UNIFESP

^IIIAssisting physician and physician in charge of the Systemic Sclerosis Outpatient Clinic of the Rheumatology Department of UNIFESP

Correspondence to

ABSTRACT

OBJECTIVES: The objective of this study was to investigate the dynamic behavior of the blood flow of the microvascular circulation of the fingertips before and after two cold stimuli (CS), using Laser Doppler Imaging with different intensities in patients with systemic sclerosis (SSc) and in healthy individuals.

PATIENTS AND METHODS: Fourteen SSc patients (51.2 ± 5.5 years) with Raynaud's phenomenon and 12 healthy controls (44.8 ± 9.0 years) were included in this study. Two CS protocols (submersion of the hands in water at 10 ºC or 15 ºC for 1 minute) were performed on the same day. Mean fingertip blood flow (FBF) of four digits of the left hand was measured using LDI (Moor LDI-VR, Moor Instruments) at baseline and at 1, 4, 10, 25, and 40 minutes after CS.

RESULTS: Baseline blood flow was significantly lower in both CS protocols in SSc patients when compared to controls (312.9 ± 102.7 vs 465.4 ± 135.4 PU, P = 0.006 at 15 ºC; 305.2 ± 121.0 vs 437.9 ± 119.8 PU; P = 0.01 at 10 ºC). In the control group, a significant decrease in FBF after CS, when compared to baseline, was observed 1 minute (P = 0.001) after CS at 15 ºC and at 1 (P = 0.005) and 25 minutes (P = 0.001) after CS at 10 ºC. In SSc patients, a significant decrease in FBF was observed in both CS protocols at 1, 4, and 10 minutes (P < 0.000; P = 0.002; P = 0.014, after CS at 15 ºC; P < 0.000; P = 0.004; P = 0.001, after CS at 10 ºC).

CONCLUSIONS: Laser Doppler Imaging showed lower baseline fingertip perfusion and further reduction after CS in SSc patients compared to controls. Quantification of fingertip blood flow by LDI may be useful in the longitudinal monitoring of the disease status and therapeutic interventions in SSc.

Keywords: systemic sclerosis, Raynaud's phenomenon, microcirculation, Laser Doppler Imaging.

INTRODUCTION

Systemic sclerosis (SSc) is an autoimmune disease of unknown etiology characterized, clinically, by vascular involvement and fibrotic changes of the skin and internal organs.¹ The early detection of peripheral vascular changes, characterized by structural and functional abnormalities in small blood vessels and microcirculation, is possible in the majority of the cases.

Raynaud's phenomenon(RyP), seen asthe firstmanifestation in 70% of the patients and in up to 95% of the cases during the evolution of the disease, is the most evident and earlier clinical manifestation of vascular involvement.² Raynaud's phenomenon is defined as an abnormal vasoconstriction in response to cold, causing recurring episodes of spasm of digital arteries, arterioles, and cutaneous arteriovenous shunts. It is characterized by typical color changes of the extremities, especially hands and feet, that classically have three phases: pallor, cyanosis, and rubor. Structural changes of the microcirculation, endothelial damage and dysfunction, and platelet activation, which usually make vasospastic events more severe, are present in RyP secondary to SSc, or diseases of the same spectrum.³ Marked reduction in blood flow, and even complete occlusion of the vessel, can be seen. All those changes can lead to chronic tissue hypoxia and irreversible tissue damage with recurring ulcers, scars, and, in more severe cases, gangrene and amputation of the extremities.⁴

Accurate methods for the evaluation and quantification of the changes present in the microcirculation in patients with RyP and SSc, may it be to study the pathophysiology of the disease or to differentiate patients with secondary RyP from those with primary RyP, have to be developed.⁵ Besides, with the advent of new treatment modalities for SSc and associated RyP, objective and reproducible methods to evaluate the response to therapy are increasingly more necessary. Those methods could be associated, or not, with acute ischemia provocation tests - immersion of the extremities in cold water or insufflation of pneumatic devices.^6,7 Currently, a method considered the gold-standard to evaluate the blood flow in the microcirculation in patients with primary RyP or SSc does not exist. Laser Doppler flowmettrics, a non-invasive method that provides quantitative evaluation of the blood flow in the microcirculation, is based on the Doppler effect of beams reflected by moving blood cells.⁸ Conventional Laser Doppler evaluates only a small circumscribed point of the skin each moment, and great signal variability among different areas represents an important limitation, making it a poorly reproducible method, limiting its use in therapeutic assays.^9,10 Laser Doppler Imaging (LDI) is based on a relatively recent technique that allows direct measurements of the blood flow of the cutaneous microcirculation over a wider area and not only in a single point. It also has the advantage of not requiring direct contact with the skin surface, eliminating changes secondary to pressure, or artifacts caused by movements.^11,12 Due to the precision and reproducibility, LDI is considered a promising method for the dynamic evaluation of the blood flow in RyP and in patients with SSc.¹³

In the present study, the response of the microcirculation to two cold stimuli of different intensities in healthy controls and in patients with SSc was evaluated. Our intention was to evaluate the dynamic behavior of the digital blood flow in healthy individuals and SSc patients.

PATIENTS AND METHODS

A transversal study with patients with the diagnosis of SSc, according to the ACR classification,¹⁴ who also had RyP and periungueal capillaroscopy with SD (scleroderma pattern) microangiopathy,¹⁵ from the Systemic Sclerosis Outpatient Clinic of HSP-UNIFESP, was undertaken. Healthy controls, paired by gender and age, were selected among the employees of Hospital São Paulo. Exclusion criteria were: smoking, diabetes, severe hypertension (diastolic blood pressure > 105 mmHg), or patients with clinically significant central vasculopathy and/or peripheral venous vasculopathy. Any vasodilators used by patients were discontinued three days before the tests. All patients should be older than 18 years and they had to sign an informed consent approved by the Ethics Commission of UNIFESP under protocol #1,298/06.

After an adaptation period (rest for one hour in a room the with temperature of 24 ± 1º C, with their hands most of the time in the axillary region to keep them warm), patients and controls underwent two consecutive protocols of cold stimuli on the same day with a minimal inter-test interval of 60 minutes. The specific conditions of both protocols were as follows:

Part I: Assessment of vasoreactivity of the cutaneous microcirculation in the presence of a cold stimulus at 15º C

A - Assessment of the baseline blood flow of the cutaneous microcirculation by Laser Doppler Imaging. The left hand of all individuals was placed on a flat surface (at the level of the heart), and the blood flow on the dorsal region of the distal phalange of four fingers (except the thumb) of the left hand was evaluated in basal conditions by Laser Doppler Imaging (Moor LDI, Moor Instruments, Axminster, UK). This device uses a low intensity helium-neon red laser beam with 633 nm with penetration in the skin surface of approximately 1 mm. The light beam is directed to the selected area of the skin by a mirror located at 40 cm from the skin surface. All images were captured at a rate of 4 pixels/millisecond, with acquisition time of 3 minutes and 15 seconds for each image. The mean blood flow of the selected area was determined with the help of the MoorLDI V5.2 software and expressed in arbitrary perfusion units (PU) in relation to an internal standard calibration of the device. The mean blood flow of the four fingertips (FBF) was considered for analysis.

B - Cold stimulus (CS). Cold stimulus was applied by immersion of both hands in water at 15º C for 60 seconds (UNITEMP 116, Fanem, Brazil).

C - Monitoring the blood flow in the cutaneous microcirculation by Laser Doppler Imaging after the Cs. The FBF of the dorsum of the four fingers (except the thumb) of the left hand was measured continuously, as described, up to 25 minutes after the CS. The blood flow measured at 1, 4, 10, and 25 minutes after CS was considered for analysis.

Part II: Assessment of the vasoreactivity of the cutaneous microcirculation in the presence of a cold stimulus of 10º C

The protocol described in Part I was repeated, observing a minimal interval of 60 minutes between both CS. The main difference relied on the CS, which consisted on immersion in water at 10º C for 60 seconds. The blood flow in the cutaneous microcirculation was monitored up to 40 minutes after the CS and measured at 1, 4, 10, 25, and 40 minutes after the CS, represented another difference in relation to part I.

Statistical analysis

To better detect the dynamic changes in FBF secondary to the CS, the percentage variation in post-CS FBF in relation to pre-CS FBF, called ∆FBF, was used, in which:

Results were expressed as mean ± standard deviation. The Student t test was used for intergroup analyses, while ANOVA for repeated measurements was used to compare the groups along time. A levelof significance of 0.05 was adopted in all the analyses.

RESULTS

Fourteen SSc patients, with mean age of 51.2 years (ranging from 41 to 60 years), and 12 healthy controls, mean age of 44.8 years (ranging from 33 to 54 years), were included in the study. Five patients (35.7%) had limited cutaneous disease and nine (64.3%) had diffuse cutaneous disease (Table 1). The duration of the disease in patients with the limited cutaneous type was 10.5 ± 4.2 years and the onset of RyP was 12.2 ± 5.1 years ago. In patients with the diffuse cutaneous type, the duration of the disease was 8.3 ± 5.6 years and the onset of RyP was 7.3 ± 6.4 years ago. Ten patients were being treated with vasodilators (calcium channel blockers, captopril, and losartan).

Basal FBF was significantly lower in SSc patients than in the controls in both CS protocols (312.9 ± 102.7 versus 465.4 ± 135.4 PU, P = 0.006, before CS at 15º C; 305.2 ± 121.0 versus 437.9 ± 119.8 PU, P = 0.01, before CS at 10º C) (Figure 1). Baseline FBF in healthy controls did not differ between the first and second CS (P = 0.341) or in SSc patients (P = 0.679), confirming the reproducibility of the method. The mean blood flow in the fingertips was significantly lower in SSc patients when compared to the control group at all times after CS at 15º C (P = 0.005, 1 minute after Cs; P = 0.009, 4 minutes after CS; P = 0.001, 10 minutes after CS; and P = 0.016, 25 minutes after CS), and at 1, 4, and 10 minutes after CS at 10º C ( P < 0.001; P = 0.008; and P = 0.002, respectively) (Table 2).

An abrupt fall in FBF was observed 1 minute after both CS in SSc patients and in the control group, followed by a gradual recovery of the blood flow, which was delayed in SSc patients (Figure 2). In healthy controls, a significant reduction in FBF was observed after CS at 15º C, when compared to baseline levels, 1 minute after CS (P = 0.001). This group showed fast recovery of FBF and statistically significant differences among basal FBF and FBF 4, 10, and 25 minutes after CS at 15º C (P = 0.055; P = 0.55; and P = 1.00, respectively) were not observed. At 10º C, significant differences were observed in post-CS FBF at 1 and 25 min when compared to baseline levels (P = 0.005; P = 0.001, respectively) in the control group. In SSc patients, a significant reduction was observed in FBF 1, 4, and 10 minutes after CS at 15º C (P < 0.000; P = 0.002; and P = 0.014, respectively) and 10º C (P < 0.000; P = 0.004; P = 0.001, respectively). A recovery in FBF was observed only 25 minutes after CS (P = 0.146 at 15º C; P = 0.25 at 10º C) (Figure 2). The magnitude of the reduction in FBF (∆CS-FBF) was greater in SSc patients than in healthy controls 1 minute after CS at 10º C (-72.5 ± 63% versus -35.3 ± 15%) (Table 2).

DISCUSSION

In the present study, we undertook a novel evaluation of the dynamic behavior of the blood flow in the microcirculation of the digits, before and after two cold stimuli of different intensities, by comparing the LDI of patients with RyP secondary to SSc and healthy individuals. The FBF of patients with systemic sclerosis was considerably lower than in healthy controls. As expected, a significant reduction in FBF was observed in both groups after CS, both at 15º C and 10º C, and this reduction was more marked in SSc patients. Healthy controls showed fast recovery in FBF, especially after CS at 15º C (observed 4 minutes after CS); the same was not true for SSc patients, who had delayed recovery of FBF. The recovery curve after CS at 15º C was capable to better characterize both groups.

The findings of lower baseline FBF levels in SSc patients confirm prior studies that reported conspicuous morphological changes, devascularization, and reduction in blood flow in the small vessels and microcirculation of those individuals.^16,17 The greater reduction in FBF observed in the majority of SSc patients might be due to changes in blood flow dynamics that are worsened by unfavorable conditions secondary to structural changes in the microcirculation observed in those individuals. The vasoreactivity of the cutaneous microcirculation is, usually, a protective response to heat loss.After a cold stimulus, SSc patients show a knowingly exacerbated response to acute ischemia, with sudden increase in vascular tonus, marked reduction of the blood flow, and even possible closure of digital arterioles.^18,19

The consistent and objective reproduction, in clinical practice, of the vasospastic events in patients with primary or secondary RyP is difficult. Several methods for the objective measurement of the severity of cold vasoreactivity in each patient with RyP, such as thermography, digital systolic blood pressure, fingertip lacticemy, plethysmography, conventional Laser Doppler, and LDI, are used.^5,12,19-23 Thermography evaluates, indirectly, the temperature of the skin of a specific area. Digital systolic blood pressure is useful to evaluate the blood flow of the digital artery; however, it does not evaluate the microcirculation. Plethysmography evaluates changes in blood volume in specific segments and it seems to be a poorly reproducible method.⁵ On the other hand, LDI allows the evaluation of larger areas of the cutaneous microcirculation, and its results are more reproducible and representative when compared to conventional Laser Doppler. Clark et al.¹⁰ used LDI to evaluate the blood flow on the back of the hands in patients with primary RyP and diffuse cutaneous SSc, limited to the room temperature of 23º C and 30º C. The authors observed significant differences in blood flow among the four study groups, especially when evaluating the maximal difference in blood flow among the different fingers of the same hand at a room temperature of 23º C. Note that the most expressive differences were observed in patients with limited cutaneous SSc and smaller differences in healthy controls.

Laser Doppler Imaging was also used to assess the therapeutic response to topical glyceryl trinitrate in patients with primary RyP and in SSc patients²⁴. An increase in digital blood flow was observed in all patients after applying glyceryl trinitrate when compared to placebo. More recently, when evaluating the long-term treatment with intravenous N-acetylcysteine in RyP secondary to SSc, Salsano et al.²⁵ observed a significant increase in global perfusion of the hands on LDI after treatment. Those studies indicate that Laser Doppler Imaging is a powerful tool in the quantification of the blood flow in the microcirculation and objective monitoring of the therapeutic response in patients with RyP and SSc. However, until now, very few studies have evaluated the vasoreactivity of cutaneous microcirculation in response to a cold stimulus in patients with RyP and/or SSc using this method.^12,26 Picart et al.²⁶ evaluated digital perfusion before and after cold stimuli (cold plate at 5º C for 8 minutes) with LDI in eight patients with SSc, ten with primary RyP, and seven healthy controls. Similar to the results of the present study, SSc patients showed lower baseline blood flow and marked decrease after the cold stimulus when compared to healthy controls.

Different protocols of cold stimuli, such as immersion of both hands or one hand in water at different temperatures (ice until 20º C) and total body cooling are used for the objective evaluation of vasoreactivity in patients with RyP.^27-30 In our study, we performed two protocols aiming at standardizing CS test with LDI and quantify, objectively, changes in blood flow in the microcirculation related to the pathophysiology of RyP. To this end, we attempted to evaluate which CS would Laser Doppler Imaging in patients with systemic sclerosis be better to distinguish healthy individuals from patients with RyP secondary to SSc. Cold stimulus at 15º C for 1 minute was used by O'Reilly et al.,³⁰ and it was considered adequate to evaluate blood flow recovery curves in the presence of a CS. Our group has used the cold stimulus at 10º C, in association with lacticemy test of the fingertips, in several studies, showing that it is capable of differentiating patients with secondary RyP, primary RyP, and healthy controls.^21-23 However, CS at 10º C is more uncomfortable and potentially more damaging for patients with RyP. The present study demonstrated that very intense or prolonged CS is not necessary to produce vasospasm or distinguish patients with secondary RyP from healthy individuals by LDI. Besides, based on the results of the present study, CS at 10º C also seemed to cause severe vasoconstriction, even in healthy individuals, therefore hindering the discrimination between patients and healthy controls.

One should not forget that measurements of peripheral blood flow can be influenced be a series of factors, such as low environmental temperature, length of acclimatization, smoking, hormonal changes, use of vasodilators or vasoconstrictors, and stress.^31,32 In our study, we tried to minimize the influence of extrinsic factors by applying a homogenous protocol, consisting on individual adjustment in a calm environment with controlled temperature, exclusion of smokers, and discontinuation of vasodilators three days before the tests, to all participants.

In conclusion, the present study showed significant lower levels of basal FBF in SSc patients when compared to healthy controls. A significant reduction in FBF was observed in both groups after CS, especially 1 minute after CS in both protocols. Cold stimulus at 15º C differentiated better both groups. Quantification of FBF associated with a CS could, therefore, be useful in the evaluation for therapeutic assays and longitudinal follow-up of patients with RyP secondary to SSc. Prospective studies with a greater number of SSc patients and patients with primary RyP could better define the role of LDI in the diagnosis and monitoring of those patients.

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Quantification of basal digital blood flow and after cold stimulus by laser doppler imaging in patients with systemic sclerosis

Marcelo José Uchoa Corrêa^I; Sandro F Perazzio^I; Luís Eduardo Coelho Andrade^II; Cristiane Kayser^III

Publication Dates

• Publication in this collection
  17 May 2010
• Date of issue
  Apr 2010

History

• Received
  17 Mar 2009
• Accepted
  24 Feb 2010