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. Author manuscript; available in PMC: 2014 Feb 1.
Published in final edited form as: Exp Eye Res. 2012 Nov 24;107:37–43. doi: 10.1016/j.exer.2012.11.003

Pilocarpine-induced Flare is Physiological Rather than Pathological

Thomas F Freddo 1,2, Nathan Neville 3, Haiyan Gong 2
PMCID: PMC3556229  NIHMSID: NIHMS424451  PMID: 23187102

Introduction

Increases in the total protein content of aqueous humor result in light scattering. This scattering, when seen clinically with a slit-lamp, is commonly referred to as “flare”. The presence of flare is generally taken as a sign of intraocular inflammation. It is well documented that miotics give rise to flare (Wessley, 1900; Seidel, 1920; Stocker, 1947; Abraham, 1959; Mori, et al., 1992; Freddo, et al., 2006). Wessley, in 1900, was the first to report this phenomenon after administering drops of the muscarinic miotic pilocarpine. Later investigators, examining pilocarpine-induced flare, have even referred to the phenomenon as miotic iridocyclitis (Abraham, 1959).

Contemporary investigators, examining the mechanisms underlying this flare, have used well-established methods, including fluorophotometry and laser cell-flare meter (Mori, et al, 1992). But these methods are unable to directly examine events in the posterior chamber of the eye in-vivo. As such, events in the posterior chamber had to be inferred using a computational model of blood-aqueous barrier (BAB) kinetics. In the model available until the mid-1990’s, the small amount of plasma-derived protein present in aqueous humor was presumed to be a part of aqueous humor as it was secreted (Raviola, 1977; Bill, 1986). This opinion prevailed notwithstanding numerous tracer studies concluding that plasma-derived proteins did not cross the tight junctions of the ciliary epithelium or the iris vascular endothelium. (Raviola, 1977; Freddo and Raviola, 1982). With the assumption that plasma-derived proteins were a part of aqueous as it was secreted, the model provided only a single possible explanation for the presence of flare in the anterior chamber. The explanation was that BAB permeability had increased in response to topical pilocarpine. Hence, using the model of the BAB available to them, Mori, et al., (1992) reached the only conclusion possible; a single drop of pilocarpine produces a transient, dose-dependent increase in BAB permeability – a breakdown of the BAB.

In a series of studies that have redefined the model of how the BAB functions, it has been shown that the plasma-derived proteins present in the aqueous humor of the anterior chamber bypass the posterior chamber and diffuse into the anterior chamber directly (Freddo et al, 1990; Barsotti, et al., 1992; Freddo, 2001, Bert, et al., 2006). This diffusional path begins in the ciliary body stroma and delivers plasma-derived proteins to a reservoir in the iris stroma, entering from the iris root. From this reservoir, these proteins gradually diffuse into the aqueous humor of the anterior chamber, since there is no epithelium on the anterior surface of the iris. But these proteins are prevented from diffusing through the posterior surface of the iris, into the posterior chamber, by tight junctions between the cells of the posterior pigmented epithelium of the iris (Freddo, 1984). Diffusion posteriorly, through the pupil, is prevented by the one-way valve created by apposition of the pupil with the anterior lens capsule, combined with the continuous forward flow of aqueous through that aperture.

From these several studies redefining the model of the BAB, it is clear that the plasma-derived protein measured in an aliquot of aqueous humor obtained from the anterior chamber is not a part of the aqueous humor as it is secreted. Instead, this protein is added to the aqueous in the anterior chamber from the ciliary body, via the iris stroma. As such, aqueous humor production and the entry of plasma-derived proteins into the aqueous humor are semi-independent variables. This leaves open the possibility that aqueous humor plasma-protein concentrations could vary, and thus clinical “flare” could occur, without compromise of the tight junctions that constitute the anatomically defined elements of the BAB.

We have documented a method that permits in-vivo evaluation of BAB kinetics in the posterior chamber using magnetic resonance imaging (MRI). Using this method we have documented that commonly used intravenous contrast agents distribute within the ocular tissues in a pattern similar to that found using intravascular surrogates for plasma-protein, such as horseradish peroxidase (Kolodny, et al., 1996; Bert, et al., 2006).

Using these methods, we reassessed the effects of topical pilocarpine on BAB permeability in the normal human eye (Freddo, et al., 2006). Sixty minutes after topical administration of one drop of 3% pilocarpine, (the time at which pilocarpine-related flare is reported to peak, Mori, et al., 1992) sequential MRI images showed that ciliary body contrast intensity increased rapidly and an increasing contrast signal, of similar magnitude, was subsequently seen in the anterior chamber of both treated and untreated eyes. During this same period, however, the baseline contrast level observed in the posterior chamber remained constant and never enhanced in either eye. These direct studies of the posterior chamber documented that, contrary to previous conclusions, pilocarpine-induced “flare” was not the result of ciliary epithelial barrier breakdown. Unfortunately, the MRI technique was not sensitive enough to conclusively show that pilocarpine did not increase the permeability of the iris vasculature.

The combination of a redefined BAB model (Freddo, 2001) and evidence suggesting topical pilocarpine does not produce an increase in permeability at the ciliary body barrier in vivo in humans (Freddo, et al., 2006), suggested that further investigation into the effects of topical pilocarpine on the iris vasculature was warranted. The aim of this study was to examine the rabbit ciliary epithelium and iris vasculature, with and without application of topical pilocarpine, to determine if BAB permeability was altered.

Materials and Methods

Nine pigmented rabbits (Myrtle’s Rabbitry, Tennessee) of either sex, weighing 2.0 to 2.5 kg were used in accordance with the Institutional Animal Care and Use Committee at Boston University and in accord with the ARVO Resolution on the Use of Animals in Research. Pigmented rabbits were used because the light irides of albino animals reflect light that can confound measurements of anterior chamber flare using laser Flare/Cell meters.

Pupil size and anterior chamber flare in rabbit eyes treated with topical pilocarpine

For these studies four of the nine pigmented rabbits were used. The rabbits were sedated with 1.0 to 2.0 mg/lb body weight acepromazine maleate i.m. injection. Baseline flare readings (photons/msec) were taken using a KOWA-FC-1000 Flare-Cell meter (Kowa Optimed, San Jose, CA). Horizontal pupil diameter (mm) of both eyes was then measured using a millimeter ruler under constant ambient illumination. One eye then received a single standard drop of 3% pilocarpine-HCl solution (Bausch & Lomb Pharmaceuticals, Tampa, FL). Prior studies have shown that peak levels of flare occur one hour after instillation of a single drop of 3% pilocarpine (Mori et al, 1992). One hour after pilocarpine instillation, flare values (photons/msec) and pupil sizes for both eyes were again measured.

Ciliary body and iris vasculature permeability analysis

One hour after pilocarpine instillation and following flare and pupil size measurements, three of these four rabbits received an intravenous injection of 250 mg/kg body weight horseradish peroxidase (HRP- Type II, Sigma Chemical Co., St Louis, MO). The HRP tracer was allowed to circulate for 3 minutes and the animals were subsequently euthanized with an intravenous injection of greater than 120 mg/kg of pentobarbital sodium (Euthasol; Virbac AH Co., Fort Worth, TX). To verify that a transient increase in iris vascular permeability had not occurred during the hour we waited for peak flare response, the fourth animal was injected with HRP immediately after a noticeable horizontal pupil diameter difference between the eyes was observed (~20 min) and sacrificed three minutes after the intravenous injection of HRP, without documenting flare levels.

Both eyes of all four animals were quickly enucleated and cut through the equator. The vitreous and lens of each eye were carefully removed. The anterior segment was cut into quadrants and the uvea was gently dissected from the corneoscleral tunic. Radial wedges of iris and ciliary body were embedded in 4% agar and 150 μm sections were made with a Smith-Farquhar tissue chopper (DuPont Sorvall, Wilmington, DE). Sections were reacted for demonstration of HRP as previously described (Raviola, 1974) and then post-fixed with 2% osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA) in 1.5% potassium ferrocyanide in deionized water for 2 hours. The sections were then dehydrated in a series of ethanols and embedded in an Epon-Araldite mixture (EMS, Fort Washington, PA). Thick sections (3 μm) were cut, stained with 0.1% toluidine blue, and either examined using an Olympus BX-40 photomicroscope, or further prepared into ultra-thin (90nm) sections for electron microscopic examination (Philips 300: Eindoven, The Netherlands).

Light micrographs of HRP distribution in the iris and ciliary body were taken at objective magnifications of 10X, 20X, and 40X. For electron microscopy, HRP-reacted ultra-thin sections of iris and ciliary body were stained with uranyl acetate and then examined for HRP distribution. Particular attention was given to the permeability characteristics of the tight junctions of the non-pigmented ciliary epithelium and the iris vascular endothelium.

Iris stromal protein reservoir experiments

One hour after pilocarpine instillation and following flare and pupil size measurements, the five remaining animals were immediately sacrificed with an intravenous injection of euthasol. Both eyes of each animal were quickly enucleated and cut through the equator. The vitreous and lens of each eye were carefully removed. The anterior segment was cut into quadrants centered on the pupil. The iris tissue in two quadrants was carefully dissected away from the ciliary body. To minimize the influence of the fenestrated vessels of the iridial processes of the rabbit, upon the outcome of the experiments, only the portion of the iris closer to the pupil than the iridial processes was sampled to analyze the total elutable protein of the iris. The other two quadrants of each eye were placed in Carnoy’s fixative and prepared for immunohistochemical evaluation of albumin distribution.

Immunohistochemistry -albumin distribution in iris, ciliary body and cornea

The samples fixed in Carnoy’s were dehydrated with a series of alcohols and chloroform, and embedded in paraffin, as described by Gong, et al. (1997). Five μm paraffin sections were cut and mounted onto microscope slides. Sections were deparaffinized, rehydrated with PBS and treated with 0.1% Triton X-100 in PBS for 1 minute at room temperature, followed by a PBS rinse. The sections were incubated for 20 minutes with 4% non-fat milk in PBS, at room temperature, and then incubated with fluorescein isothiocyanate (FITC) conjugated sheep anti-rabbit albumin antibody (1:200, Bethyl Laboratories Inc., Montgomery, TX) overnight in PBS-moistened chambers at 4°C. The sections were rinsed 6 times with PBS and then washed with PBS for 5 minutes. Sections were counter-stained with Topro3 (1:1000, Invitrogen, Carlsbad, California) for 8 minutes to visualize cell nuclei and washed three times with PBS. The sections were coverslipped and sealed with nail polish.

Substitution of sheep anti-rabbit albumin antibody with PBS or anti-sheep IgG (1:200 in PBS) were used as controls. All sections were examined using a Carl Zeiss Laser Scanning System LSM 510. Images were taken under 10X objective. FITC channel settings remained constant. Raw data was exported through the use of a Zeiss LSM Image Browser.

Total elutable protein analysis of iris tissue

The protocol for total elutable protein analysis was modified from Gong, et al. (1997). From wedges of anterior uvea, the portion of iris closer to the pupil than the iridial processes of the iris was gently cut away with a sharp blade. The wet weight of this portion of each iris sample was taken (mg) and each specimen was then placed into 1 ml of phosphate buffered saline (PBS). Samples were put on a rotary shaker for 4 hours at 4°C. The eluant was transferred into a 1.5 ml centrifuge tube and centrifuged at 12,000×G for 30 minutes. The resulting supernatant was used to determine the total soluble protein. Total protein analysis of eluant was completed using a BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL). For each iris sample, four spectrophotometer readings were averaged. Samples from paired pilocarpine-treated and untreated irides were compared and were reported as the average μg albumin/wet weight (mg) per eye.

Statistical analysis

Paired or unpaired t-tests with either equal or unequal variance were performed to determine statistical significance. Statistical significance was achieved if the p-value was <0.05. Data is presented as mean ± standard deviation.

Results

Pupil size and anterior chamber flare in rabbit eyes treated with topical pilocarpine

Pupil diameter and flare values of are summarized in Table 1. Physiological anisocoria was not seen in any animals. The average horizontal pupil diameter of the pilocarpine eyes (4.7±0.66) one hour after pilocarpine instillation, was significantly smaller than in control eyes (7.2±0.5) (p≪0.001).

Table 1.

Summary of Pupil Size and Aqueous Flare

PupilSize Baseline Flare Flare Post-Pilo
Rabbit Pilo Control Pilo Control Pilo Control
1 4 6.5 7 5 21 7.5
2 5 7 14.1 10 28 5.9
3 4 7 8.1 9.3 18.9 8.9
4 6 7 *** *** *** ***
5 5 7 4.5 4 31.7 3.9
6 5 7.5 6.2 6 28.6 4.2
7 4 7 5.4 5.8 17.3 2.9
8 5 8 10.5 8.8 29.2 7.8
9 4.5 8 3.6 * 24.5 5.1
Mean±SD 4.7±0.6 7.2±0.5 7.43±3.5 7.0±2.3 24.9±5.3 5.87±2.3
P-value 0.000004 0.19 0.00004
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***

No flare measurements taken, animal euthanized twenty-two minutes after instillation of pilocarpine.

Average baseline flare readings taken prior to instillation of pilocarpine did not differ between the experimental and control eyes (p=0.19) (Table 1). Baseline flare readings in the control eyes (7.0±2.34 photons/ms) did not change relative to the fellow eye after administration of pilocarpine (5.87±2.28) (p= 0.11). One hour after receiving pilocarpine there was a significant increase in flare levels in eyes treated with pilocarpine (24.9±5.3 photon/ms) compared to the baseline flare readings (7.43±3.5 photon/ms) (p=0.00006).

Ciliary body and iris vasculature permeability analysis

Light microscopy

In both experimental and control eyes, iris blood vessels showed no histological evidence of HRP leakage (Figure 1A,B). Light microscopy demonstrated the presence of HRP within the lumen of the iris blood vessels in both the treated and the untreated eyes. In pilocarpine-treated eyes, HRP remained confined to the lumens of the vessels and was not found in the surrounding iris stroma as would have been expected if pilocarpine had increased vascular permeability. No histological evidence of increased iris blood vessel permeability was observed. HRP was observed in the ciliary body stroma of both the pilocarpine and control eyes (Fig. 2). To confirm that the measured increase in flare did not arise from a transient increase in blood-aqueous barrier permeability that had resolved by the one hour time-point, one animal was sacrificed at the first noticeable decrease in pupil size in the eye treated with pilocarpine (Table 1).

Fig. 1.

Fig. 1

Fig. 1

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(A) Light micrograph of 3% pilocarpine-treated iris stroma stained with 0.1% toluidine blue. HRP (brown) is demonstrated within iris vasculature, but is not seen in the surrounding iris stroma. (B) Light micrograph image of control iris stroma stained with 0.1% toluidine blue. (AC) anterior chamber, (PC) posterior chamber.

Fig. 2.

Fig. 2

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Light micrograph of 3% pilocarpine ciliary body stained with 0.1% toluidine blue. HRP (brown) is demonstrated within the stroma of the ciliary body (arrows).

Electron microscopy

Electron microscopy confirmed what was seen by light microscopy. There was no evidence of HRP leakage across the iris vascular endothelium or ciliary epithelium in either pilocarpine-treated or control eyes (Figs 3A, 4A). No alterations of tight junctions were found in either the ciliary epithelium or the iris vascular endothelium (Figs 3B, 4B).

Fig. 3.

Fig. 3

Fig. 3

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Electron micrographs of 3% pilocarpine-treated iris vasculature. (A)HRP is found within vessel lumen, without leakage into surrounding stroma. (B)Tight junctions of the iris vascular endothelium appear intact (arrow). HRP is seen within the blood vessel lumen.

Fig. 4.

Fig. 4

Fig. 4

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Electron micrograph of 3% pilocarpine-treated ciliary body demonstrating HRP distribution. (A) HRP is seen between pigmented ciliary epithelial cells (thick arrow) and along the shared apical surfaces of the pigmented and non-pigmented ciliary epithelial layer (arrowhead). Movement between adjacent non-pigmented ciliary epithelial cells toward the posterior chamber is blocked by the tight junction of the nonpigmented ciliary epithelium (thin arrows). (B) Higher magnification demonstrates HRP between pigmented ciliary epithelial cells and along the apical surfaces between the two epithelial layers (thin arrow). Intercellular cleft between adjacent non-pigmented cells is stopped by a tight junction, leaving remainder of interecellular cleft free of HRP (thick arrow). (NPCE) nonpigmented ciliary epithelium, (PCE) pigmented ciliary epithelium.

Immunohistochemistry -albumin distribution in iris, ciliary body and cornea

Confocal imaging showed that albumin was widely distributed in the anterior uvea. As shown in Figure 5, albumin was readily found in the stromas of the iris, ciliary body, and cornea of both the control and the pilocarpine treated samples. In the iris stroma, diffuse albumin staining was found from the iris root to the pupillary margin, most notably in the control samples. Albumin staining patterns of the cornea were consistent with findings of Gong et al, (1997); albumin was seen in the corneal stroma, with greater amounts in the anterior than the posterior stroma. Anti-Sheep IgG controls showed essentially no staining in the cornea and iris stroma (Fig. 5C). The PBS controls showed no staining of any structure (not shown).

Fig. 5.

Fig. 5

Fig. 5

Fig. 5

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(A) Immunohistochemistry (IHC) of pilocarpine eye prepared with sheep anti-rabbit albumin antibody-FITC. In sections of cornea, iris and ciliary body (CB), albumin is observed in the cornea, iris stroma and ciliary body. (B) IHC of control eye prepared with sheep-rabbit albumin antibody-FITC. In sections of cornea, iris, and ciliary body, albumin is observed in the cornea, iris stroma and ciliary body. (C) IHC of tissue prepared with anti-sheep IgG (Control). No albumin staining found in cornea or iris stroma.

Total elutable protein analysis of iris tissue

Pupil size, flare values, and total elutable protein for the remaining five pigmented rabbits are summarized in Table 2. As expected, pilocarpine produced a significant increase in flare (p=0.0003) and a significant decrease in pupil size (p=0.0002) (Table 2). When pooled albumin readings from the pilocarpine-treated group and control group were compared, the amount of albumin found in the iris stroma of pilocarpine-treated eyes was significantly less than that found in those of controls (p= 0.049) (Table 2). Of additional interest, there was not a correlation between the end-point pupil size and the amount of total elutable protein within the experimental group. There was however, a correlation between the magnitude of the change in pupil size (2.8±0.57) and eluted protein levels in the pilocarpine-treated eyes (6.55±2.4) (p=0.027).

Table 2.

Summary of Total Elutable Protein

Total Elutable Protein Flare Post-Pilo Baseline Flare Pupil Size
Rabbit Pilo Control Pilo Control Pilo Control Pilo Control
1 7.67 9.27 31.7 3.9 4.5 4 5 7
2 5.43 10.07 28.6 4.2 6.2 6 5 7.5
3 2.81 3.48 17.3 2.9 5.4 5.8 4 7
4 8.7 12.1 29.2 7.8 10.5 8.8 5 8
5 8.19 21.07 24.5 5.1 3.6 * 4.5 8
Mean±SD 6.55±2.4 11.2±6.3 26.3±5.6 4.78±1.9 6.66±2.7 6.15±1.98 4.7±0.4 7.5±0.5
P-value 0.049 0.0003 0.17 0.0002
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Baseline flare values (photons/ms), before administration of pilocarpine, show no significant difference between eyes treated with 3% pilocarpine and control (p=0.17). Flare significantly increases after one hour in eyes instilled with topical 3% pilocarpine (p=0.0003). Iris tissue of pilocarpine-treated eyes show significantly less average elutable protein than control (p=0.049). Horizontal pupil size of pilocarpine-treated eyes decreased significantly one hour after instillation of pilocarpine (p=0.0002).

*

Flare-cell meter malfunction.

Discussion

In our previous study on this topic, (Freddo, et al., 2006) a single drop of 3% pilocarpine was administered to one eye of normal human subjects and both the progressive miosis and increase in flare were documented for an hour after instillation. Using a gadolinium intravenous contrast agent and high resolution MRI, we demonstrated that no increase in contrast entry into the posterior chamber occurred in experimental vs control eyes. We also addressed the issue of whether there was a short, transient, increase in ciliary epithelial permeability that occurred during the hour we waited before proceeding with the MRI. In this control subject, the MRI study was initiated just after pilocarpine installation and, again, no increase in signal intensity was measured in the posterior chamber. Gadolinium, with a molecular weight of 938 kd would, if anything, overestimate leakage of plasma-proteins across the ciliary epithelium. Unfortunately, with MRI, we could not rule out an increase in iris vascular permeability as the source of the flare.

The results of the current study strongly suggest that the flare that follows instillation of a single drop of pilocarpine does not result from an increase in the permeability of the tight junctions that constitute the anatomical equivalents of the BAB. These results confirm the conclusion of our previous study, using MRI and contrast agents in humans, demonstrating that pilocarpine does not increase ciliary epithelial permeability. But the current study also extends these findings, indicating that there is also no demonstrable increase in iris vascular permeability caused by a single drop of 3% pilocarpine.

The present studies confirm the presence of a reservoir of plasma-derived proteins within the stroma of the iris (Kuchle, et al., 1995). The source of this protein is not the iris vasculature but the result of diffusion from the fenestrated vessels of the ciliary body (Bert, et al., 2006). This reservoir is prevented from diffusing backward into the posterior chamber by the tight junctions of the posterior epithelium of the iris (Freddo, 1984).

But if pilocarpine does not produce flare by increasing the permeability of the ciliary epithelium, or the iris vascular endothelium, what is the source of the flare? The results of the current studies demonstrate that the amount of diffusible protein remaining in the iris stroma following instillation of pilocarpine was significantly less than in control eyes. Moreover, there was a correlation between the percent change in pupil size (i.e. strength of miotic response) and eluted protein levels in the pilocarpine-treated eyes.

Because the unique anatomy of the rabbit iris brings fenestrated vessels out into extended iridial processes in this species, we only sampled the portion of the iris that was devoid of the additional source of protein diffusion into the iris stroma from these processes. As such, direct comparisons between the total amount of flare and the amount of protein lost from the iris stroma were not possible, either biochemically or histochemically.

Clearly pilocarpine produces flare following a single topical administration (Wessley, 1900; Seidel, 1920; Stocker, 1947; Abraham, 1959; Mori, et al., 1992; Freddo, et al., 2006). But it has also been reported to produce constant faint flare with regular use and to aggravate the flare response in inflamed eyes (Kanski and Holborn, 1968). In the present studies we directly assessed only the effect of a single drop of pilocarpine. Nonetheless, in this scenario, it is clear that pilocarpine does not cause flare by increasing the permeability of the tight junctions that constitute the anatomical equivalents of the BAB. Instead, it appears more likely that the dramatic thinning of the iris stroma that attends miosis extrudes some of the constantly present reservoir of plasma-protein from the stroma into the anterior chamber. Given the correlation between levels of eluted protein and the strength of the miosis, as measured by percent change in pupil size, this extrusion of protein into the aqueous humor is more likely the cause of the transient flare.

A modest additional contribution to pilocarpine-induced flare may arise from the fact that pilocarpine reduces aqueous humor formation rate (Nagataki and Brubaker, 1982). In such cases, if the protein pathway kept delivering plasma-derived proteins at a constant rate, while aqueous secretion was reduced, protein concentration would increase due to a concentration effect.

Still another another potentially significant contribution may arise from the fact that by contracting the ciliary muscle, pilocarpine blocks the passage of colloids from the anterior chamber into the suprachoroidal space and the posterior uvea, (Bill and Walinder, 1966). In the absence of pilocarpine one would expect that a portion of the protein normally leaked from the fenestrated capillaries of the ciliary body would move posteriorly, away from the anterior chamber. But, in the presence of pilocarpine, virtually all of this protein would be redirected forward toward the anterior chamber. This mechanism is especially intriguing because it could account for the chronic faint flare that accompanies repeated use of pilocarpine. Indeed, it could also account for the augmentation of flare in inflamed eyes as well. Future studies will be required to explore these hypotheses.

In conclusion, the major implication of the present work is that, contrary to widely accepted belief, not all clinically observed flare is pathological. To our knowledge this is a new concept. Thus, certain forms of flare may have a physiological rather than a pathological basis.

Highlights.

This study demonstrates that:

  • Pilocarpine-induced flare does not result from blood-aqueous barrier breakdown.

  • The flare results from miosis-induced extrusion of protein from the iris stroma.

  • This provides proof of principle that not all clinical flare is pathological.

Acknowledgments

This work was supported by NEI Grant #EY-13825 (TFF), by a Summer Research Fellowship from Fight for Sight, Inc. to NN and by an unrestricted grant to the Department of Ophthalmology at Boston University from The Massachusetts Lions Eye Research Fund, Inc. Excellent technical assistance was provided by Rozanne Richman, M.S. The authors would also like to thank Alcon Pharmaceutical Company for their generous loan of a KOWA FC-1000 Flare Cell Meter.

Footnotes

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