Measurement of multiple microcirculatory parameters in human nasal mucosa using laser-doppler velocimetry

MICROVASCULAR

RESEARCH

38, 175-185 (1989)

Measurement of Multiple Microcirculatory Human Nasal Mucosa Using Laser-Doppler HOWARD M. DRUCE, MICHAEL A. KALINER, ROBERT F. BONNER

Parameters in Velocimetry

DAVID RAMOS, AND

Division of Allergy and Immunology, Department of Internal Medicine, St. Louis University School of Medicine, 1402 S. Grand Boulevard, St. Louis, Missouri 63104; and Allergic Diseases Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases and Biomedical Engineering and Instrumentation Branch, Division of Research Services, National Institutes of Health, Bethesda, Maryland 20892 Received December 8, 1988 LDV has been modified to measure four microcirculatory responses in human nasal mucosa. Resting nasal blood flow was measured in I15 observations in 23 nonatopic subjects and 111 observations in 21 atopic subjects with allergic nasal disease. Other parameters measured concurrently were the number density of moving red blood cells (RBC), mean RBC speed, and flow pulsatility. Challenges with aerosolized buffered saline or water had no significant effect on any parameter. By contrast, nasal application of cY-adrenergic agonists, oxymetazoline and phenylephrine, produced significant dose-dependent reductions in flow without any significant change in RBC number density. These results suggest a selective a-agonist effect on resistance vessels but not on capacitance vessels. Topical cholinergic stimulation with methacholine selectively reduced the RBC number density without affecting other parameters. These modifications of LDV may prove useful in analyzing nasal responses to provocation and determining the sites of action of vasoactive agents on the microcirculation. 0 1989 Academic Press. Inc.

INTRODUCTION LDV has been widely used to measure microcirculatory responses in a variety of human tissues, e.g., skin, muscle, and nasal mucosa (Rodgers et al., 1984; Tahmoush et al., 1983; Druce et al., 1984). Data presented to date have been limited to the single parameter of local microcirculatory blood flow (QLDV) (Bonner et al., 1981a). The NIH prototype LDV instrument has been adapted to measure the volume of RBC within a cubic millimeter of tissue; this parameter is referred to as the RBC number density (m) (Bonner et al., 1981a,b). From these two measured parameters, two other physiological meaningful factors, mean RBC speed (V,,,) and flow pulsatility [P(Q)], may be derived. This paper describes these parameters as they relate to human nasal mucosa and their response to pharmacologic provocation.

175 0026-2862189 $3.00 Copyright Q 1989 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in U.S.A.

176

DRUCE

MATERIALS

ET

AL.

AND METHODS

Subjects

Forty-four human subjects were studied after informed consent had been obtained. Twenty-three of the subjects were nonatopic (10 male and 13 female) and 21 had allergic nasal disease (11 male and 10 female). All of the subjects were between the ages of 18 and 45 years; they were nonsmokers, had no systemic disease, were not pregnant, and had not taken any medications for the 24 hr preceding the study. The nonatopic subjects had no history of rhinitis or asthma nor did they have a family history of allergies. They were recruited from a pool of normal healthy volunteers and were on no medications. Physical examination was normal and prick-skin testing with a battery of inhalant and food antigens was negative. The atopic subjects were selected on the basis of seasonal rhinitis (along with asthma in some of the patients) and positive reactions to skin tests with the inhalant antigens consistent with their clinical history. Positive reactions were defined as at least a 3 + reaction to the relevant antigen, together with a 3 + histamine control and negative saline control. All subjects were studied at times of the year when they were symptom-free and were not using any medication which might have an effect on the nose for at least 1 month preceding the study. In addition they were taking no regular medications which might have systemic effects. LDV Parameters

The principles and theory of LDV have been explained in detail elsewhere (Bonner et al., 1981a,b). In brief, LDV uses the coherent properties of laser light to monitor the mean velocity and number density of moving RBCs in a cubic-millimeter sample of tissue. The mean Doppler shift multiplied by the fraction of light that is Doppler shifted is proportional to the QLDV. QLDV is obtained by analog computation of the normalized first moment of the power spectrum of the photocurrent associated with the detection of diffusively backscattered light from the tissue. The fraction of light that is Doppler shifted is proportional to the volume of flowing blood If(Vblood)] or number density of flowing RBCs within the tissue (m). Two further parameters have been derived from Q and m (Table 1). I’,.,,,, is proportional to the mean RBC speed and is computed by dividing Q by m. Pulsatility of flow P(Q) = (peak flow - trough flow)/mean flow at any given time point. Measurement

of Laser-Doppler

Parameters

The configuration of the LDV instrument and nasal fiberoptic probe have described previously (Druce et al., 1984). Output of nasal blood flow and number density were recorded on a chart recorder. Values under baseline ditions and 3 min after challenge were subsequently analyzed using the Statistical Program (SAS Institute, Cary, NC). Challenge

been RBC conSAS

Materials

The sources of nasal challenge materials were as follows: sterile pyrogen-free water and 0.9% sodium chloride solution (Abbott Laboratories, Chicago, IL): 0.05% oxymetazoline hydrochloride solution (Schering Corp., Kenilworth, NJ):

LASER-DOPPLER

177

VELOCIMETRY

1% phenylephrine hydrochloride (Winthrop Laboratories, New York, NY); and methacholine hydrochloride (Sigma Chemical Co., St. Louis, MO). Concentrations of phenylephrine and methacholine hydrochloride were diluted from stock with sterile water, buffered to pH 5.3, and resterilized by passage over a 0.22pm filter (Millipore Corporation, Bedford, MA). Challenge Procedure Nasal challenges were performed under direct vision by spraying the solutions with the use of an oxygen-driven atomizer head, as described previously (Druce et al., 1984). For the challenges, we used 0.12 + 0.02 ml of solution delivered beyond the anterior vestibule of the nose in two sprays administered in immediate succession. LDV parameters Q and V,,, were monitored continuously during these procedures. To measure LDV parameters, a 3-mm-diameter probe was placed lightly on the anterior aspect of the inferior turbinate under direct vision and held in place by a clamp attached to a set of audio headphones. The subjects remained in a comfortably seated position and were instructed to remain still during the procedure. The subjects were initially aware of the presence of the probe but became accustomed to it within several minutes. In no instance was the probe described as uncomfortable. After the LDV probe was gently placed on the subjects’ inferior turbinate, stable baseline readings of blood flow and volume were achieved within 2 to 5 min. Subsequently, all challenges were performed without disturbing the probe. Up to seven sequential challenges were made at 5-min intervals. For challenge protocols employing saline or water, 0.12 ml of solution were sequentially introduced at 5-min intervals. For oxymetazoline challenges, two sequential 60-mcg doses were given at 5-min intervals. Phenylephrine was administered at 5-min intervals in a sequence of increasing doses (120, 240, 600, 900, and 1200 mcg). Each individual study with phenylephrine or oxymetazoline was terminated when the blood flow had fallen to 50% of baseline or, if this fall did not occur, when all sequential doses in the series had been given. Methacholine was also given in increasing doses (0.006, 0.06, 0.6, 6, and 12 mg) at 5min intervals. Each subject studied in the methacholine protocol received each of these doses. In a preliminary trial of phenylephrine and oxymetazoline effects, very large doses of these agents (2 x -10 x ) did not significantly decrease blood flow below an average of 60% of the baseline flow. In order to minimize the exposure of the subjects to high doses and to avoid systemic effects, our dose regimen of increasing doses was terminated if a 50% reduction of baseline flow was observed. Each subject underwent up to 10 separate nasal challenge procedures and as many as 17 measurements of resting parameters. Each challenge procedure lasted approximately 1 hr, and challenges were not performed on the same subject more frequently than once per week. The same protocol was not repeated more than one time on a given subject. The protocol describing these studies was approved by the clinical research subpanel of the National Institute of Allergy and Infectious Diseases. Statistics Data were compared

using the two-tailed

Student’s

t test for summary

data.

178

DRUCE ET AL.

RESULTS The baseline microcirculatory parameters defined above (Table 1) were calculated on 226 independent measurements. These data are shown in Table 2, with the mean, standard deviation, and standard error of the mean for each parameter. Comparisons of these measurements were computed separately for the nonatopic and atopic groups. Using the t test for summary data to compare these results, we found no statistically significant difference between the nonatopic and the atopic populations. Similar atopic/nonatopic comparisons based on sex of subject, side of nose under observation, ambient room temperature, or relative humidity revealed no differences in the mean baseline measurements of the LDV parameters. This observation is important in that the subjects within a given group may be considered as one population when, for example, the parameters are varied through the introduction of drugs or neurohormones. Since we observed no microcirculatory parameter in baseline data which discriminated between the population groups, we proceeded with the nasal challenges with the nebulized solutions. To determine whether a nebulized solution, of itself, would influence nasal blood flow, we began with saline as the first challenge substance. Table 3 shows the effect of saline on the LDV parameters. The prechallenge values in the table refer to the data for the subjects given the saline challenge, not the total pool of baseline data. There was no difference in response between nonatopic and atopic subjects, hence the results are shown pooled. The four parameters show no statistical difference between the pre- and the postchallenge values, as demonstrated by the two-tailed t test. Although no significant changes in the LDV parameters were noted in response to individual challenges (Table 3), analysis of the cumulative effect of up to seven sequential saline challenges showed a slight but significant reduction in nasal blood flow (Q) of 15% (Druce et al., 1984). This prompted us to do a series of challenges with sterile water. These experiments demonstrated that all these parameters remain constant after serial nasal challenges with nebulized saline or water. Hence, any changes produced by neurohormonal challenge may be attributed to the neurohormone in question and not the diluent.

TABLE LASER-DOPPLER

Q m V ml5

p(Q)

1 PARAMETERS

Volumetric blood flow (ml of blood/100 g of tissue/mm): obtained from LDV RBC flow W by Q = k’ E/m Number density of moving RBCs: fraction of signal that is Doppler shifted f(m) = 2m Mean root mean square of RBC speed: determined from mean Doppler shift Pulsatility of flow: P(Q) = [Q(systole) - Q(diastole)l/Q(mean)

Note. W = k f(m) V,,,, the laser-Doppler fraction of signal that is Doppler shifted.

flow parameter. k, k’ = optical constants. f(m) =

LASER-WPPLER

TABLE BASELINE

MICROCIRCULATORY

PARAMETERS

179

VELOCIMETRY

OF HUMAN

ATOPIC

STATUS

2 NASAL

MLJCOSA,

STRATIFIED

Nonatopic (n = 115)

Q m V PG,

BY NONATOPIC

OR

OF &EJECT

Mean

SD

SEM

Atopic (n = 111) Mean SD SEM

39.1

11.4

1.1

37.6

11.4

1.1

17.2

5.3

0.5

17.6

4.8

0.5

0.10 1.0

0.01 0.1

0.9 0.10

0.01 0.1

Volumetric blood flow (ml blood/100 g tissue/min) Number density of moving RBC Mean Flow pulsatility RBC speed

0.16 2.3

2.3 0.17

Challenges with Neurohormonal Agents Oxymetazoline hydrochloride. This is a widely prescribed nasal decongestant; its effect in reducing nasal blood flow (Q) has been described previously (Druce et al., 1984). The (u-2 adrenergic agonist properties of this drug predominate, exceeding its a-l agonist potency and affinity (Starke, 1981). The effects of oxymetazoline on the parameter of flow (Q) have been shown previously to be quantitatively similar in nonatopic and atopic subjects (Druce et al., 1984). Table 4 shows the effects of oxymetazoline on LDV parameters. Each subject studied was given oxymetazoline until mucosal blood flow was reduced by 50% or more. The mean cumulative dose given was 87 mcg. While the flow (Q) and speed (V,,,) were reduced, the pulsatility [P(Q)] was significantly increased (from 0.15 to 0.25), and the RBC number density (m) was unchanged. Phenylephrine hydrochloride. Phenylephrine hydrochloride is a potent, shortacting nasal decongestant with the pharmacologic properties of an a-1 adrenergic agonist (Weiner, 1980). The effects of phenylephrine on the LDV parameters are also shown in Table 4. The mean cumulative dose of phenylephrine given was 710 mcg. As was seen with oxymetazoine, the parameters Q and V,,, were reduced (to 24.9 and 1.5) and, conversely, the pulsatility [P(Q)] was increased almost twofold (from 0.17 to 0.30). The data for phenylephrine challenge were also analyzed by plotting the change

EFFECT

OF SALINE

OR STERILE

WATER

TABLE 3 LDV PARAMETERS,

ON

PRE-

Saline (n = 42)

Q in Vrlns

P(Q)

Pre Post Pre Post Pre Post Pre Post

AND

POSTCHALLENGE

Water (n = 38)

Mean

SD

SEM

Mean

SD

SEM

40.9 37.8 16.6 17.7 2.4 2.1 0.14 0.17

10.2 13.1 4.8 4.0 1.3 1.2 0.08 0.11

1.6 2.0 0.7 0.6 0.2 0.2 0.01 0.02

41.2 40.6 17.0 18.3 2.4 2.2 0.15 0.17

10.6 9.9 4.8 3.7 1.3 0.6 0.08 0.10

1.7 1.6 0.8 0.6 0.2 0.1 0.01 0.02

180

DRUCE TABLE

EFFECT

OF THE a-ADRENERGIC

ET AL. 4

AGONISTS PHENYLEPHRINE AND PRE- AND POSTCHALLENGE

Phenylephrine

OXYMETAZOLINE

(n = 160)

ON LDV

Oxymetazoline

Mean

SD

SEM

Mean

PARAMETERS,

(n = 51) SD -__--

SEM

&

Pre Post

40.9 24.9

10.0 13.7

0.8 1.1*

39.2 27.9

11.8 14.6

1.7 2.0*

In

Pre Post

18.7 18.3

5.7 4.9

0.4 0.4

16.0 16.1

5.0 5.0

0.7 0.7

V rnla

Pre Post

2.2 1.4

0.9 1.1

0.1 0.1*

2.4 1.7

1.1 1.1

0.2 0.2*

P(Q)

Pre Post

0.17 0.30

0.09 0.28

0.01 0.02*

0.15 0.25

0.08 0.30

0.01 0.04*

* P < 0.001

in each LDV parameter against the dose of drug associated with that change (Fig. 1). From these figures, the relationships between the microcirculatory parameters and cumulative phenylephrine dose may be seen. As the dosage was increased, blood flow (Q) and speed (V,,,) both decreased, whereas pulsatility [Z’(Q)] continued to increase until a dose of almost 1000 mcg of phenylephrine was reached. Blood volume (m) was unaffected by phenylephrine. Methacholine hydrochloride. Cholinergic stimulation with methacholine produced a pattern of changes on LDV parameters which differed from the pattern observed with a-adrenergic agents (Table 5). The mean cumulative dose of methacholine given was 15 mg. Blood flow (Q) and pulsatility [P(Q)] were not significantly changed; the RBC number density (m) was significantly reduced to 13.4; and the speed (V,.,,) was slightly increased, but this latter change failed to reach statistical significance at P = 0.06. DISCUSSION To demonstrate the action of circulating and local hormones and neurohormones in health and disease, a tissue may be perturbed by topical application of these agents in order for the responses to be studied. In addition to its obvious importance as an indicator of nasal disease, such as allergic rhinitis, the nasal mucosa may be useful as a model system to study mucosal responses. The nasal mucosa represents a highly vascular tissue that is easily accessible to topical agents which can be applied under direct vision. The nasal mucosal vasculature, which functions as a temperature control and a humidifier for inspired air (Cauna, 1982), demonstrates significant reactivity that can be monitored by LDV. In this paper, we have extended our LDV observations (Druce et al., 1984) to look at three novel LDV parameters. To achieve this, a novel circuit to measure m and V,,, was developed (Bonner et al., 1981a). The derivation of units for LDV parameters is of importance (Table 1). The flow parameter (Q) has been detailed elsewhere (Druce et al., 1984). Units for m are relative. Since the output of the circuit has not been calibrated to units of tissue hematocrit, units represent the output of the system as a percentage

LASER-DOPPLER

PHEh’YLEPHRlNE

CUMUIATIVE

VELOCIMETRY

DOSE $4G)

PHENYLEPHRINE 0

CUMUlArmE

DOSE

(pG)

DOSE

(/iG)

0.8

0.4

0.2

0

lOCiI

PHENYLEPHRINE

2ooa CUMUlATlVE

3ooo DOSE

4000

0

(CLG)

1000

PHENYLEPHRINE

2ow CUMULATlVE

3ooo

FIG. 1. The four microcirculatory parameters measured are each plotted against the cumulative dose of phenylephrine in micrograms administered up to the point of measurement. (A) Blood flow (Q) in ml blood/100 g tissue/min. (B) The number density of flowing RBCs (m) in arbitrary units. (C) The mean RBC speed (V,,,) in arbitrary units. (D) The pulsatility in flow P(Q). TABLE EFFECT

OF METHACHOLINE

5 ON

LDV

PARAMETERS

(n = 27)

Q m

Vmls P(Q)

Pre Post Pre Post Pre Post Pre Post

* P < 0.01. t P = 0.06.

Mean

SD

SEM

40.8 40.9 16.1 13.4 2.5 3.0 0.15 0.15

11.0 11.9 4.8 3.1 1.3 1.1 0.08 0.18

2.1 2.3 0.9 0.6* 0.3 0.2t 0.02 0.03

182

DRUCE

ET

AL.

of the light that is Doppler shifted. Similarly, the value of V,,, is expressed in arbitrary, although absolute, units. The conversion factor from mean frequency to mean speed has been described (Bonner et al., 1981a). Pulsatility of flow [P(Q)] represents a ratio and thus is not expressed in units. The intersubject variation for all four parameters measured (Table 2) is wide, as reflected in the relatively large standard deviations. This range which was noted previously with respect to nasal blood flow (Druce et al., 1984) has also been observed in xenon-133 washout (Bende, 1983) and hydrogen clearance studies (Tanimoto et al., 1983) as well as in measurements of nasal airway resistance (Cole et al., 1980; Solomon and McLean, 1983). In our previous study, no correlation between blood flow and ambient temperature, relative humidity, age, disease state, pulse rate, arterial blood pressure, or the particular side of the nose under observation was seen. We concluded that the variability reflects normal variations in resting sympathetic tone to the blood vessels. Table 2 shows that in addition to the mean values of the LDV parameters, the degree of variability is also similar in nonatopic and atopic individuals. In the next set of experiments, we investigated the effects of aerosolized saline sprayed into the nose. This was done to test the stability of the LDV system in the presence of a pharmacologically inert substance, which might affect the LDV parameters by a physical process. The saline, which was sterile and at room temperature, contained no preservatives and had been buffered to pH 5.3. The changes in the LDV parameters were minimal after each challenge. In our earlier study of volumetric blood flow (Druce et ul., 1984), we noted that the cumulative effect of up to seven sequential challenges with saline caused a maximal reduction of 15% in nasal blood flow, which is not apparent when each challenge is analyzed separately (Table 3). This observation prompted us to do a further series of studies employing sterile water under the same physical conditions and experimental protocol. The results from these studies (Table 3) indicate that introducing a nebulized solution of water produces no significant changes in any of the parameters. The LDV parameters obtained may be useful in selectively analyzing components of the microcirculation. The local vessels may be divided into two major categories-resistance and capacitance. The compliance of the resistance vessels (arterioles) dampens the large pulsatility of the arterial blood flow as it passes through the microcirculation. Increases in the tone of these vessels would reduce the perfusion pressure in the capillary exchange vessels and/or reduce the number of capillaries perfused. Both of these effects would result in reduced capillary exchange. In some tissues, including the nasal mucosa, a significant proportion of blood flow is diverted through arteriovenous shunts. In the nasal mucosa, high-speed arteriovenous shunt flow provides efficient convection heating that makes possible the rapid warming and humidification of inspired air before its entry into the lungs. The data shown herein, in which reductions in Q and V,,, occur without corresponding changes in m and P(Q) after application of (Yadrenergic agonists, also suggests that the baseline flow which is perturbed by these agents is dominated by arteriovenous shunt flow. It is interesting to note that whereas phenylephrine and oxymetazoline have different spectra of a-adrenoceptor agonist activity (Starke, 1981), their effects on the nasal microcirculation were similar. This similarity has also been observed

LASER-DOPPLER

VELOCIMETRY

183

in animal studies (Ichimura and Jackson, 1984). It is likely that the nasal vasculature responds to stimulation by both types of receptors, and an aggregate result is being observed. Selective antagonists might be useful in this system in uncovering differential effects. It has not been determined how much active drug reaches the receptor, due to a host of variable conditions in nasal mucosa (Chien and Chang, 1987). However, for nonpolar water-soluble low-molecular-weight molecules such as phenylephrine and oxymetazoline, 100% of the dose deposited on the mucosa should be absorbed. It is not possible to estimate how much is degraded before reaching the receptors. In one study in which nasal mucosal blood flow was measured by the xenon-133 washout method (Andersson and Bende, 1984), it was reported that oxymetazoline reduced blood flow whereas phenylephrine did not, and that yohimbine (an a-2 adrenergic antagonist) pretreatment blocked the effects of oxymetazoline. However, only single doses of agonists and antagonists were used, and only five or six observations were made in each subject group. Studies with dose-response curves would further elucidate this. An in vivo study on dog nasal blood vessels demonstrated the presence of both a-l and -2 postsynaptic receptors, both of which produced smooth muscle contraction upon stimulation (Ichimura and Jackson, 1984.) Any significant alteration in the change in the RBC number density would reflect changes in the capacitance, rather than the resistance, vessels. Thus, m may increase either with the number density of capillaries being perfused locally, or the degree of engorgement of the venous sinusoids. a-agonist challenge does not significantly change m, suggesting that the number density of capillaries being perfused does not change. The reduction in Q, but not in m, represents the physiologic effect of the a-adrenergic agents on the arteriovenous shunts, which are morphologically highly adapted to control flow (Cauna, 1970; Nagai et al., 1983). This is in agreement with animal data (Maim, 1977). One might infer that the constant blood volume suggests that the venous sinusoids are unaffected by a-adrenergic stimulation and that induced tissue volume changes are due to resorption of interstitial fluid. It is possible that the LDV system does not observe vessels deep enough into the mucosa to reflect the more deeply situated capacitance sinusoids. However, this is unlikely because these studies were performed on normal, noncongested mucosa. Another possibility is that the blood in the large capacitance sinusoids is stagnant and hence moving so slowly so that no signal could be detected from these vessels. It remains undetermined whether reduction in arteriovenous shunt flow due to cY-adrenergic effects causes secondary changes in the venous sinusoids. Methacholine hydrochloride is a stable analog of acetylcholine and is widely employed to mimic parasympathetic nerve stimulation. Previous studies have demonstrated that parasympathetic stimulation of human nasal mucosa causes an increased volume of nasal secretions, but no significant change in nasal airway resistance, which is an indirect reflection of the tone of the capacitance vessels (Borum, 1979). Similar data have been reported with animal studies (Anggard, 1974). The effects of methacholine in the LDV system were studied to determine whether any change occurred in microcirculatory parameters. The methacholine doses administered did not affect heart rate or systemic blood pressure and, hence, we conclude that the changes observed in the nasal mucosa are most likely due to local microvascular effects.

184

DRUCE

ET

AL.

Responses to methacholine challenge demonstrated a reduction in RBC number density, but not in flow, mean speed, or pulsatility (Table 5). These results are consistent with a microvascular effect solely on the capacitance vessels of the mucosa. This conclusion is,in agreement with a study using histochemical staining which showed that few cholinergic receptors are associated with the resistance vessels (Anggard, 1974) and that cholinergic nerve fibers end primarily in glands (Normura and Matsuura, 1972). In summary, measurement of several additional laser-Doppler parameters, under baseline conditions and after topical provocation challenge, has produced new information regarding the nasal mucosal microcirculation beyond that obtained by measuring nasal blood flow. This methodology may prove useful in assessing the microcirculatory responses of other tissues. Also, the availability of paired LDV instruments might permit differentiation of local from systemic effects, and enhance the study of nasal reflexes. ACKNOWLEDGMENT The authors thank Margaret Smith for excellent secretarial support.

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